The positive energy and immune status of ME-supplemented rabbit does were reflected in their productive and reproductive performance. ME-supplemented rabbit does had better colostrum and milk yield and composition as well as enhanced fertility traits. Colostrum is a special type of milk formed during the last days of pregnancy and the first few days after birth. The main components of colostrum in rabbits are proteins and fats. Thus, the nutritional requirements for these components are elevated, specifically toward the end of pregnancy and the early stages of lactation. For example, the coefficients of variation in total protein concentration reach their maximum during the early stages of lactation (5–26%), and immunoglobulins account for most of the total protein in colostrum [47]. As reported in previous studies [38,47], we found that ME improved colostrum protein and fat contents due to its high amino acid and FA concentrations. The improved protein content and different immunoglobulin (IgG, IgA, and IgM) in colostrum reflects an increased availability of amino acids for immunoglobulin synthesis [48] and improves the passive transfer of immunoglobulin to the mammary gland. Moreover, some FAs identified in ME have been shown to improve immunoglobulin contents in colostrum; for example, colostrum IgG concentrations were increased in animals fed an n-3 PUFA-rich diet [9].

Supplementation of ME also improved both milk yield and milk composition, specifically protein and fat content. Given that the ME-treated rabbit does had increased BWs and blood metabolites (higher glucose and lower fat metabolites) than the control rabbit does during the energy-consuming lactation period, it can be said that ME played a role in supporting energy requirements and modulating metabolism in favor of milk production without negative effects on energy balance status. In fact, feeding either Moringa leaf or ME has been shown to increase milk production in many farm animals, including rabbits [49,50]. Moringa contains plenty of nutrients, such as amino acids, FAs, and vitamins, which are precursors for milk synthesis [51]. Moreover, it contains phytosterols, phytoestrogens that can boost both lactogenesis and mammogenesis [18]. Moringa is rich in phytosterols, such as sitosterol, stigmasterol, and campesterol, which are precursors for hormones. These phytochemicals, along with high amino acid contents, can enhance estrogen excretion, stimulating mammogenesis and milk production [20].

In the present study, all ME treatments significantly increased the concentrations of milk USFAs, MUFAs (C16:1n-7 and C18:1n-9), omega-6 PUFAs (conjugated linoleic acid [CLA] and AA), omega-3 PUFAs (linoleic acid [LA], alpha-linolenic acid, EPA, and DHA) compared to the C group. As identified from the ME FA profile, ME has considerable concentrations of omega-3 PUFAs (EPA and DHA) and omega-6 PUFAs (LA, gammalinolenic acid, and dihomo-gamma-linolenic acid), which are implicated in PUFA longchain biosynthetic pathways. LA is the main precursor for omega-6 FA pathways; it can be used as a precursor for longer omega-6 PUFAs via the effects of elongase and desaturase enzymes [46]. This pathway seems to be enhanced in this study, as ME-treated rabbit does had higher concentrations of longer omega-6 PUFAs (CLA and AA) in their milk. The same trend was observed for omega-3 PUFAs, and omega-3 EPA can be used as a precursor for omega-3 DHA biosynthesis [16,17]. Overall, ME supplementation to rabbit does during lactation improved FA bioavailability to the mammary gland and activated FA biosynthetic pathways, leading to different FA profiles in the milk of ME-supplemented rabbit does. These positive effects might also be related to the improved energy status and the availability of energy-yielding nutrients, such as glucose, during lactation. It has been confirmed that the FA elongation process can be stopped if glucose, the acetyl-CoA source, is not adequately available [2,4].

Results of the present study confirmed the superior reproductive performance of the ME-supplemented rabbit does, which had better fertility and pregnancy outcomes than the non-supplemented (C) rabbit does. The positive effect of ME on the energy status of rabbit does is needed to achieve better reproductive performance. Several studies reported that higher BWs of rabbit does around mating time and early pregnancy are associated with better artificial insemination outputs [52,53]. In addition to the positive role of ME on body weight and metabolism, the unique FA composition of ME might have other positive effects. As reported in other studies, FA-rich diets can improve fertility and pregnancy outcomes in rabbits [15,46]. In fact, FAs are an important element for many reproductive events through various modes of action. Numerous FAs can positively influence reproduction by altering the ovarian follicles and corpus luteum function via improved energy status and increasing precursor levels for the synthesis of reproductive steroids and prostaglandins [54]. They also improve the competence of oocytes and boost embryo development [55] and pregnancy and fetal development [56]. For example, PUFAs, such as EPA and DHA can improve the quality of embryos by decreasing apoptosis rates and improving cell membrane integrity [15,57]. Moreover, omega-3 PUFAs are associated with low PGF2α production in uterine and placental tissues, decreasing the susceptibility to abortion and/or preterm parturition [58].

In this study, the ME-treated rabbit does had better pregnancy outcomes (litter sizes and weights at birth and weaning) than the control rabbit does. The developing fetuses and the offspring in the first days of lactation are totally dependent on their dams to provide the nutrition required for growth and development. Thus, such positive effects of ME on pregnancy outcomes might begin early and be related to improved oocyte quality, embryo development, and implantation during the early stages of pregnancy by providing specific FAs and/or energy sources. Additionally, during the later stages of pregnancy, most fetal lipids are delivered from maternal circulation through the placenta and are obtained from the diet or fat lipolysis [59], constituting the fat deposits on which the survival of the newborn depends. In this study, no decreases in dams body weight were observed in the ME-treated rabbit does. This finding leads us to speculate that ME was effective to support metabolism during pregnancy, providing the lipid/FA requirements for fetuses without depleting the dams' fat stores. Because rabbit kits are altricial, the improved pregnancy outcomes may also be related to the improved growth rate and viability of litters after birth [18]. ME improved both colostrum immunoglobulin contents and milk production and composition and increased concentrations of some FAs that are important for boosting litter growth and health. Focusing on the important FAs for rabbit kit growth and development, higher EPA and DHA contents in milk were associated with the development of the nervous system because these FAs are used for membrane phospholipid synthesis [15,18]. Other FAs, such as AA, have been shown to boost the immune system and health status of kits [14].

### **5. Conclusions**

In the present study, ME FAs served as a good source of energy and provided functional FAs for the ideal completion of specific physiological events related to milk production and reproduction. These effects were reflected in the improved metabolism, immune status, milk production, and reproductive performance of rabbit does at different reproductive stages. These effects were observed in rabbits supplemented with free and encapsulated forms of ME. However, the nano-encapsulated form allowed for an 80% reduction (10 mg/kg BW) in the optimal dose (50 mg/kg BW) without affecting the treatment efficiency, highlighting the importance of nano-encapsulation for improving FA bioavailability.

**Author Contributions:** Conceptualization, N.I.E.-D., N.M.H. and A.G.E.; methodology, N.I.E.-D. and N.M.H.; validation, N.I.E.-D. and N.M.H.; formal analysis, N.M.H.; investigation, N.I.E.-D. and N.M.H.; data curation, N.I.E.-D. and N.M.H.; writing—original draft preparation, N.I.E.-D. and N.M.H.; writing—review and editing, N.M.H. and supervision, Z.R.A.-E., A.G.E. and N.M.H. and All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The experimental procedures were previously assessed and approved by Alexandria University-Institutional Animal Care and Use Committee (AU-IACUC, AU 08 21 07 26 2 82).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available because of privacy.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

### *Article* **Chitosan Nanoparticles Containing Lipoic Acid with Antioxidant Properties as a Potential Nutritional Supplement**

**Katrin Quester 1, Sarahí Rodríguez-González 2, Laura González-Dávalos 2, Carlos Lozano-Flores 2, Adriana González-Gallardo 3, Santino J. Zapiain-Merino 1, Armando Shimada 2, Ofelia Mora 2 and Rafael Vazquez-Duhalt 1,\***

**Simple Summary:** Alfa-lipoic acid (ALA) is an important antioxidant that could be added to animal feed as a nutritional supplement. To improve its stability in the digestive system, ALA was encapsulated in chitosan nanoparticles. The nanoparticles containing ALA were stable in stomach-like conditions and were able to cross the intestinal barrier. Chitosan-based nanoparticles seem to be an attractive administration method for antioxidants, or other sensible additives, in food.

**Abstract:** The addition of the antioxidant α-lipoic acid (ALA) to a balanced diet might be crucial for the prevention of comorbidities such as cardiovascular diseases, diabetes, and obesity. Due to its low half-life and instability under stomach-like conditions, α-lipoic acid was encapsulated into chitosan nanoparticles (Ch-NPs). The resulting chitosan nanoparticles containing 20% *w*/*w* ALA (Ch-ALA-NPs) with an average diameter of 44 nm demonstrated antioxidant activity and stability under stomach-like conditions for up to 3 h. Furthermore, fluorescent Ch-ALA-NPs were effectively internalized into 3T3-L1 fibroblasts and were able to cross the intestinal barrier, as evidenced by everted intestine in vitro experiments. Thus, chitosan-based nanoparticles seem to be an attractive administration method for antioxidants, or other sensible additives, in food.

**Keywords:** antioxidant; cell internalization; chitosan nanoparticles; everted intestine; lipoic acid

### **1. Introduction**

Reactive oxygen species (ROS) are highly reactive molecules generated as by-products of oxygen metabolism in aerobic organisms [1]. Several diseases, such as hypertension and diabetes, amongst others, are associated with ROS generation [2,3]. Aging is one of the known interrupters of the balance between ROS generation and antioxidant defenses [4], and an age-related increase in oxidative damage to DNA, proteins and lipids was shown [5], particularly in cardiac and vascular tissues [6]. To slow down this oxidation-related damage, the antioxidant vitamins A, C, and E are popular nutritional supplements. However, clinical studies have failed to confirm their benefits in the prevention of cardiovascular diseases [7–9].

α-Lipoic acid (ALA), also known as thioctic acid or 1,2-dithiolane-3-pentanoic acid, is a natural antioxidant found in plants as well as in animal cells, where it is commonly found in mitochondria and acting as co-factor for a number of enzyme complexes that are involved in energy generation. Both existing forms of ALA—the oxidized (disulfide) and

**Citation:** Quester, K.; Rodríguez-González, S.; González-Dávalos, L.; Lozano-Flores, C.; González-Gallardo, A.; Zapiain-Merino, S.J.; Shimada, A.; Mora, O.; Vazquez-Duhalt, R. Chitosan Nanoparticles Containing Lipoic Acid with Antioxidant Properties as a Potential Nutritional Supplement. *Animals* **2022**, *12*, 417. https://doi.org/ 10.3390/ani12040417

Academic Editors: Antonio Gonzalez-Bulnes and Nesreen Hashem

Received: 11 January 2022 Accepted: 27 January 2022 Published: 10 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the reduced (dithiol; dihydrolipoic acid or DHLA) forms—showed antioxidant properties, and the ALA/DHLA couple is often referred to as the "universal antioxidant" due to its ability to not only improve but also restore intrinsic antioxidant systems after quenching free radicals [10–13]. Besides its great antioxidant potential, ALA was shown to efficiently remove heavy metals from the blood stream [11,13,14]. ALA has gained considerable attention as a dietary supplement because of its antioxidant activity and considerable anti-aging, anti-inflammatory, detoxifying, cognitive, cardiovascular, anti-cancer, and neuroprotective properties [15]. Some studies indicate that ALA might play an important role in the treatment of severe diseases such as diabetes mellitus [16–18], Alzheimer's disease [19–25], obesity [26–30], multiple sclerosis [31–33], and schizophrenia [34–36].

Our research group previously demonstrated that broilers fed a diet supplemented with 40 mg/kg ALA for 7 weeks had significantly decreased liver levels of thiobarbituric acid reactive substances and hydroxyl radicals, whereas their total glutathione pools were increased compared to birds fed the same diet but without ALA [37]. We also demonstrated that the mRNA expression levels of the genes encoding four enzymes involved in energy metabolism (glutathione S-transferase theta 1 (GSTT1), oxoglutarate (alpha-ketoglutarate) dehydrogenase (OGDH), pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), and dihydrolipoamide S-succinyl transferase (DLST)), as well as those encoding sirtuins 1 and 3, showed significant decreases in ALA-treated broilers compared to untreated controls (*p* < 0.01). These results confirm that the responses of broilers to ALA were associated with the down-regulation of certain liver enzymes, especially those involved in glucose metabolism and the tricarboxylic acid (TCA) cycle [38].

Nevertheless, the amount of this antioxidant synthesized by the organism does not meet bodily needs [39] unless supplementarily administered through diet. In addition, ALA presents a low half-life and bioavailability due to hepatic degradation, reduced solubility, and instability in the stomach environment [40]. Therefore, improving ALA administration is the main challenge, and the encapsulation of ALA into biocompatible nanoparticles seems to be a promising alternative.

Chitosan is a polymer produced after deacetylation of the natural polymer chitin, which is the main component of the shells of crabs and other crustaceans. The United States Food and Drug Administration (FDA) and the European Commission, among others, have approved chitosan as safe for use in food and drugs. Chitosan has been widely used for the encapsulation of proteins and therapeutical enzymes [41–44] or as biocatalytic nanoparticles [45]. Importantly, among the biopolymers used as nanocarriers, it has attracted significant attention since it is positively charged [46]. Therefore, this polysaccharide can be customized for a wide variety of chemical and ionic side-chain reactions [44]. Chitosan nanoparticles are known to easily achieve a sustained slow release of the cargo as well as increase bioavailability and therapeutic efficiency, while being biodegradable and non-toxic [47]. The production process for nanoparticles such as chitosan NPs could be improved to gain the advantages of ease of production, high yield at low cost, and larger loading capacity [48–52].

In this study, chitosan-α-lipoic acid nanoparticles (Ch-ALA-NPs) with antioxidant activity were synthesized, and their stability under stomach-like conditions was assayed. Furthermore, the cell internalization of chitosan nanoparticles in intestinal cells and fibroblasts and the ability to cross the intestinal mucosal barrier in vitro by using the everted intestine technique were demonstrated.

### **2. Materials and Methods**

*2.1. Synthesis of Chitosan-α-Lipoic Acid Nanoparticles (Ch-ALA-NPs)*

The chitosan nanoparticles were prepared based on the protocol described by [53] with the following modifications: Chitosan obtained from the deacetylation of shrimp shell chitin was purchased from Future Foods (Tlalnepantla de Baz, Mexico). The preparation showed 90.4% deacetylation, a viscosity of 54 mPa·s in 1% acetic acid, and an average molecular weight of 290 kDa. The chitosan solution was prepared by dissolving 2.5 mg mL−1 of chitosan in 2% acetic acid while stirring at approximately 200–300 rpm overnight at room temperature, and then centrifuging for 15 min at 8000× *g* at 4 ◦C. The resulting supernatant was again centrifuged for 20 min at 11,000× *g* at 4 ◦C. Then, the supernatant was filtered through a 1.2 μm membrane, and the pH was adjusted to 4.5.

Nanoparticle synthesis was achieved using an automatic pipet system designed by our research group and equipped with an insulin syringe to control the addition of reagents at a constant rate. The reagent addition was performed at room temperature and constant agitation of approximately 800 rpm. To 5 mL of the chitosan solution, 200 μL of α-lipoic acid solution (6 mg mL−1 in 20% ethanol) were added at 0.09 mm/s. After 15 min, 1 mL of tripolyphosphate pentasodium (TPP; Sigma-Aldrich, St. Louis, MO, USA) solution (0.25 mg mL−1) was added at 0.09 mm/s and left to react for 1 h. Then, 100 μL of 2.5% glutaraldehyde (Sigma-Aldrich, USA) were added at 0.09 mm/s and again left for 1 h. Afterwards, the solution was centrifuged for 30 min at 2000× *g* at room temperature (Sorvall Legend RT centrifuge) to remove chitosan aggregates. The resulting supernatant was then ultracentrifuged for 2 h at 60,000× *g* at 4 ◦C (Beckman Coulter Optima XPN-100 ultracentrifuge). Subsequently, the pellet, containing the chitosan-α-lipoic acid nanoparticles (Ch-ALA-NPs), was resuspended in 100 mM phosphate butter (pH 6), filtered through a 0.2 μm membrane, and stored at 4 ◦C until further modification.

### *2.2. Synthesis of Chitosan-Green Fluorescent-Nanoparticles Ch-GFP-NPs*

Chitosan nanoparticles labeled by encapsulating carboxylated green fluorescent protein (GFP) were synthetized as previously reported [44]. In brief, GFP was reacted with malonic acid in the presence of carbodiimide (EDC) (N-(3-dimethyl-aminopropyl)-N ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide (NHS). The amount of malonic acid was twice the amount of free carboxylic groups on the protein surface on a molar basis, and the amounts of EDC and NHS were ten and five times the amount of malonic acid, respectively. The mixtures were allowed to react for 3 h in rotation at room temperature and then dialyzed against MES (50 mM, pH 6). The encapsulation of carboxylated GFP was performed by ionic gelation with TPP as described above.

### *2.3. Morphological Analyses of the Nanoparticles*

The hydrodynamic diameter and Zeta potential of all preparations were determined by the DLS technique on a Zetasizer NanoZS (Malvern, UK). To confirm the size and shape of the nanoparticles, high-resolution transmission electron microscopy (HR-TEM) was performed using a JEOL JEM-2010. Five μL of a 1000 times diluted sample of the nanoparticles were placed onto a carbon-coated 300 mesh copper grid (Ted Pella, Inc.; Redding, CA, USA) and then stained with phosphotungstic acid (PTA).

### *2.4. Cargo Capacity and Antioxidant Activity of the Ch-ALA-NPs*

To estimate the amount of encapsulated α-lipoic acid, HPLC analyses were performed using the supernatant after ultracentrifugation. The resulting concentration of free α-lipoic acid was subtracted from the initial α-lipoic acid concentration used for nanoparticle synthesis.

Antioxidant activity of free and Ch-ALA-NPs was determined by using the inhibition of pyrogallol autooxidation method reported by [54], with some modifications. First, 50 mM Tris-HCl buffer (pH 8.2, containing 1 mM EDTA) was oxygenized with air. Pyrogallol (2 mM) was dissolved in this oxygenized buffer. The capacity of the free ALA and Ch-ALA-NPs to inhibit the spontaneous pyrogallol oxidation in the presence of atmospheric oxygen was determined using equivalent concentrations of the nanoparticles and free α-lipoic acid and monitored in a UV-vis spectrophotometer (Lambda 25, Perkin Elmer, Waltham, MA, USA) at 420 nm.

### *2.5. Release of α-Lipoic Acid from the Ch-ALA-NPs*

The release of α-lipoic acid from the chitosan nanoparticles was determined in HCl (pH 2) at 37 ◦C for 3 h, to simulate the stomach environment. A suspension of Ch-ALA-NPs (10 mL) was added to a dialysis membrane with a molecular weight cut-off (MWCO) of 14 kDa. The dialysis membrane containing the nanoparticle suspension was transferred into 20 mL of HCl (pH 2). Under constant agitation at room temperature, 1 mL samples of HCl were measured at 330 nm at different time points to determine the corresponding amounts of released α-lipoic acid. The released ALA was quantified by using a standard curve.

### *2.6. Fluorophore Labeled Nanoparticles*

In order to monitor and quantify the nanoparticles containing α-lipoic acid in the everted intestine experiments, the nanoparticles were conjugated with a fluorophore (Fluorescein isothiocyanate, FITC), and produced as follows: One mL of ALA solution in isopropanol (100 mg/mL) was added to 35 mL solution of chitosan (2%) in acetic acid, pH 5.5. The ALA solution was added at a rate of 0.09 mm/s using the automatic pipet system designed by our research group and described above. After 15 min, 250 μL of a solution of FITC in methanol (10 mg/mL) were added at a rate of 0.03 mm/s. Then, 7 mL of 0.25 % TPP were added at a rate of 0.09 mm/s, and the mixture was shaken for 1 h to produce Ch-ALA-FITC-NPs by ionic gelation. Finally, 1 mL of glutaraldehyde (2.5%) was added with the same system and shaken for 1 h. The nanoparticle suspension was centrifuged at 3500 rpm for 30 min to eliminate the bigger particles, and the supernatant was then centrifuged at 60,000× *g* for 2 h. The pellet containing the Ch-ALA-FITC-NPs was resuspended in Milli Q water.

### *2.7. Internalization of Ch-GFP-NPs into Fibroblasts 3T3-L1*

Fibroblast 3T3-L1 (CL-173, ATCC) cells (1 × 105) were cultivated in DMEM containing 10% fetal bovine serum, 1% penicillium and 1% streptomycin in 6-well plates (Corning, NY, USA) containing microscope glass slides and incubated at 37 ◦C and 5% CO2. Once they reached a confluence of 80–85%, Ch-GFP-NPs were added in different concentrations (5, 10, 20 and 50 μL mL−1) to the medium and incubated for 24 and 48 h. Afterward, the glass slides, on which cells adhered, were washed with PBS, stained for 15 min with MitoTracker Red CMXRos (Invitrogen, Waltham, MA, USA), and fixed with 4% PFA. Cell nuclei were stained with 4 ,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA). Cells were then analyzed with a confocal laser scanning microscope (LSM 780, Carl Zeiss Inc., Oberkochen, Germany) using a DPSS laser with emission at 561 nm and a HeNe laser with emission at 633 nm. Obtained images were analyzed using the software ZEN Blue Edition (Zeiss, Jena, Germany) and ImageJ (NIH, Bethesda, MD, USA).

The 3D reconstruction was performed with Amira software (Thermo Fisher Scientific, Waltham, MA, USA) by visualizing, processing, and analyzing data obtained by 780 LSM confocal microscope (Zeiss, Jena, Germany). Nuclei were seen in blue and mitochondria in red, while nanoparticles were in green; the observed signal was processed by the isosurface and volume rendering module. This visualization was carried out using immersive reality within the Cave Automatic Virtual Environment (CAVE) of the LAVIS-UNAM laboratory.

### *2.8. Intestine Barrier Crossing of Ch-ALA-FITC-NPs by In Vitro Everted Intestine*

Intestinal tissues were taken from Wistar rats and chickens (*Gallus gallus*) (donated from a local farm), to compare NP translocation through the intestine barrier in two species by everted intestine experiments. Wistar rats were housed and used at Universidad Nacional Autónoma de México (UNAM) according to regulations of the Mexican government regarding the use of laboratory animals for research purposes (CICUAL-UNAM, NOM-062-ZOO-1999 4.2.2). Animals were sacrificed with CO2 and by cervical dislocation by trained personnel. This protocol was approved by the Institute's Research Ethics Committee (Comité de Ética en la Investigación, INB-UNAM) with register #065. Intestinal tissue was obtained from 20–21 day old male rats, and 6-month-old chickens. Twelve cm

of intestine from each rat or 10 cm from each chicken were dissected and placed in a Petri dish containing Krebs-Ringer buffer at 37 ◦C. Immediately, the eversion of the intestine was carefully realized using a Pasteur pipette. The intestinal content was removed, and the tissue was washed with the same buffer. The lower end of the intestine was closed using a nylon thread, 600 μL Krebs-Ringer buffer were added, and the other end of the intestine was closed as well. The sealed intestine was then transferred into a 15 mL Falcon tube, previously gasified with CO2, containing 50 μL/mL Ch-ALA-FITC-NPs in Krebs-Ringer buffer, and incubated for 60 min at 37 ◦C under agitation. Subsequently, the intestinal content was centrifuged for 1 min, and fluorescence of the supernatant was spectrofluorimetrically measured at an excitation of 495 nm and an emission of 519 nm. Control experiments were carried out in the same conditions but without adding nanoparticles. The percentage of nanoparticle internalization was estimated by comparing the fluorescence of the samples to the fluorescence of the initial nanoparticle solution. All experiments were performed in triplicate.

### *2.9. Statistical Analysis*

Significant differences in all determinations were analyzed by one-way ANOVA test, followed by post hoc Tukey rank test. Statistical significance was set at *p* < 0.05 and expressed as compact letter display. Statistical analysis was performed on five independent replicates using the software STATISTICA 8.0™ (StatSoft Inc., Tulsa, OK, USA).

### **3. Results**

Chitosan nanoparticles were produced by ionic gelation in the presence of ALA. Preliminary experiments to determine the maximal ALA loading in the chitosan nanoparticles were performed. A fixed amount of chitosan (12.5 mg) and different concentrations of ALA from 0.5 to 5 mg were used to produce chitosan ALA-loaded nanoparticles (Ch-ALA-NPs). The ALA content was then determined spectrophotometrically. Higher ALA concentrations than 5 mg induced precipitation and no nanoparticle formation.

The aim of this work was to produce chitosan nanoparticles as carriers for ALA. Four different preparations were synthetized: control nanoparticles without ALA (Ch-NPs), chitosan nanoparticles containing green fluorescent protein (Ch-GFP-NPs) to monitor cell internalization of nanoparticles by confocal microscopy, FITC-modified and -containing ALA particles (Ch-ALA-FITC-NPs) to evaluate intestinal barrier crossing by the everted intestine technique by fluorescence, and the ALA-containing chitosan nanoparticles (Ch-ALA-NPs) (Table 1). The nanoparticles formed with a Ch:ALA ratio of 2.5:1 showed an ALA content of 20% *w*/*w*, and this ratio was selected for further experiments.

**Table 1.** Hydrodynamic diameter and Zeta potential of different preparations of chitosan-based nanoparticles.

a,b,c,d Significant differences from four independent determinations were analyzed by one-way ANOVA test, followed by post hoc Tukey rank test. Statistical significance was set at *p* < 0.05.

Dynamic light scattering (DLS) analyses (Table 1) revealed a hydrodynamic diameter of approximately 44.1 (±20.8) nm, which was confirmed by high-resolution transmission electron microscopy (HR-TEM) (Figure 1). The nanoparticle size was half that of the nanoparticles without ALA (Ch-NPs) and could be due to a strong interaction between the carboxylic group of ALA and the amino group of glucosamine monomers of chitosan. This interaction was also supported by the reduction of zeta potential from 49 mV to 32 mV when the ALA was loaded into nanoparticles (Table 1).

**Figure 1.** HR-TEM images of synthesized Ch-ALA-NPs reveal an average diameter of 44 nm. Scale bars are 100 nm in (**A**), and 0.1 μm in (**B**).

The antioxidant activity of both free and nano-encapsulated ALA was also determined (Figure 2). The inhibition of the spontaneous oxidation of pyrogallol in the presence of atmospheric oxygen was used as the antioxidation assay. The antioxidant activity was not significantly affected by the encapsulation, Ch-ALA-NPs being slightly more efficient than free ALA, and both preparations were able to completely inhibit this reaction at the same ALA equivalents. Chitosan-only nanoparticles (Ch-NPs) showed some antioxidant activity in the oxidation of pyrogallol.

**Figure 2.** Antioxidant activity of Ch-NPs, CH-ALA-NPs and free ALA. The antioxidant activity was determined by using the inhibition of pyrogallol autooxidation method reported by Marklund and Marklund [54]. The average values from five independent experiments and the standard deviation as error bars are shown.

As mentioned above, the low half-life and bioavailability of ALA, as well as its instability in the stomach, represent disadvantages for its administration as a dietary supplement. Thus, the stability of Ch-ALA-NPs and liberation of ALA in a stomach-like environment (HCl solution at pH 2) were determined. Free ALA was measured via UV-vis spectrophotometry every 5 min for 3 h (Figure 3). A sustained release of ALA was found, but the percentage of released ALA from the NPs was very low according to the total ALA content in the NPs, implying that the Ch-ALA-NPs remained stable and intact in the stomach until further intestinal absorption. Unmodified chitosan is fully soluble at low pH. Nevertheless, the glutaraldehyde crosslinking of chitosan nanoparticles made them stable even in very acidic conditions, thus retaining the ALA inside.

**Figure 3.** ALA release from Ch-ALA-NPs in a stomach-like environment (HCl, pH 2).

Cell internalization of nanoparticles in 3T3 cells of mouse embryonic fibroblasts was assayed using synthesized fluorescent NPs containing the green fluorescent protein (Ch-GFP-NPs) (Figure 4). These fluorescent chitosan nanoparticles showed hydrodynamic diameter and zeta potential similar to non-labeled nanoparticles (Ch-NPs). A culture of 3T3 cells of mouse embryonic fibroblasts was incubated with different concentrations of Ch-GFP-NPs; after 48 h of incubation, the cells were washed three times with PBS and analyzed by confocal laser scanning microscopy (see Video S1, supplementary information). The cell nuclei were stained with DAPI, and the mitochondria with MitoTracker Red. The confocal analysis showed clear evidence of chitosan nanoparticle cell internalization.

The capacity of chitosan nanoparticles loaded with α-lipoic acid to cross the intestinal barrier was evaluated by using the everted intestine technique. To achieve this, the nanoparticles were labeled with FITC (Ch-ALA-FITC-NPs) to be monitored and quantified. Because the FITC was covalently bonded to the amino groups of glucosamine monomers of chitosan, the zeta potential was reduced to 28 mV (Table 1), but still positive. The everted intestine technique is widely used to evaluate intestinal absorption of nutrients, drugs, and toxicants [55–57]. Everted intestines from rats and chickens were incubated with Ch-ALA-FITC-NPs, and the fluorescence was measured in samples taken from both inside and outside of the everted intestine (Figure 5). Although the absorption rate was low (~0.5%/h), a significant level of fluorescence could be observed compared to the control without NP treatment.

**Figure 4.** Cell internalization of chitosan nanoparticles containing GFP into 3T3 cells of mouse embryonic fibroblasts. Ch-GFP-NPs were incubated for 48 h and then washed three times. The cell nuclei were stained with DAPI, and the mitochondria with MitoTracker Red. The nanoparticles were detected by emission at 561 nm.

**Figure 5.** Internalization through the intestinal barrier of Ch-ALA-FITC-NPs assayed in vitro by the everted intestine technique. Fluorescence in arbitrary units (AU) in the control experiments without nanoparticles and after 60 min incubation with Ch-ALA-FITC-NPs. The experiments were carried out at 37 ◦C, and average values from five independent experiments and the standard deviation as error bars are shown. Significant differences in all determinations were analyzed by one-way ANOVA test, followed by post hoc Tukey rank test. Statistical significance was set at *p* < 0.05. The meaning of a,b,c in figure is the statistical significance expressed as compact letter display.

### **4. Discussion**

α-Lipoic acid is synthesized in human and animal cells at a low rate, and thus it should be consumed as a dietary supplement. However, ALAs' instability in the stomach and low bioavailability represent challenges for efficient administration to the animal organism [40]. To overcome these obstacles, ALA was encapsulated in chitosan nanoparticles. This natural biopolymer is approved by most agencies around the world as safe for use in food and drugs, and it is known to easily achieve a sustained slow release of its cargo as well as increase bioavailability and therapeutic efficiency [47].

Chitosan-based nanoparticles containing ALA were produced (Figure 1 and Table 1). Due to the solubility of chitosan at low pH, the nanoparticles were stabilized by glutaraldehyde crosslinking. However, the glutaraldehyde molecules were covalently conjugated to the glucosamine moieties and not in free form, and thereby reducing their potential toxicity. The antioxidant activity of ALA was slightly higher when encapsulated in chitosan nanoparticles, while the NPs without ALA showed some antioxidant activity (Figure 2). The slight antioxidant capacity of chitosan is well known [58–60].

The chitosan nanoparticles were internalized by mouse embryonic fibroblasts (Figure 4 and Video S1). Chitosan nanoparticle uptake by different cells has been clearly demonstrated [61–63]. The Ch-ALA-NPs showed a positive Zeta potential of 35.7 ± 3.1 at pH of 6. The positive charge promoted the cell internalization rate in different cell lines, increasing the cellular uptake of NPs. In addition, an intracellular trafficking study indicated that positively charged NPs exhibit perinuclear localization and escape from lysosome [60].

Using the everted intestine technique, the capacity of chitosan nanoparticles containing α-lipoic acid to cross the intestinal barrier was demonstrated (Figure 5). Nanoparticles in a size range of 0.1 to 100 nm can quickly enter the digestive tract [64]. Due to their size (84 nm), the chitosan nanoparticles showed the ability to cross the mucosal barrier and interact with the subjacent absorbent epithelial tissue [65,66]. On the other hand, chitosan is a polymer that can help the absorption and increase the permeation capacity of drugs through the paracellular pathway by the reversible opening of the narrow epithelial bonds [67]. In addition, chitosan is muco-adhesive, and thus can adhere to the intestinal mucosa through ionic interactions between its positively charged amino groups and the negative charges found on the surfaces of intestinal cells, promoting intestinal absorption [68]. It is well known that chitosan nanoparticles can increase the drug concentration at the absorption sites [65,66]. In addition, it has been demonstrated that chitosan nanoparticles increase the intestinal absorption in vitro [69] of diverse bioactive compounds including, phenolic compounds [70], catechins and epigallocatechin from green tea [71], resveratrol and cumarin-6 [72].

The results obtained in the present study indicate that the administration of α-lipoic acid in the form of Ch-ALA-NPs as a supplement to a balanced diet might be beneficial for, the prevention and/or treatment of severe diseases such as cardiovascular diseases, diabetes and obesity, amongst others. ALA may play an important role in the treatment and/or prevention of diverse severe diseases including diabetes, Alzheimer's, multiple sclerosis, schizophrenia, and obesity [16,17,21,22,25–28,31,32,35].

### **5. Conclusions**

Chitosan nanoparticles containing α-lipoic acid with antioxidant activity were efficiently synthesized in a size average of 44 nm. The nanoparticles proved to be stable under stomach-like conditions for up to 3 h. Furthermore, the cell internalization of chitosan nanoparticles into fibroblasts cells was demonstrated. Finally, the nanoparticles were able to cross the intestinal barrier as confirmed by the everted intestine technique, suggesting an efficient method to supply ALA. From our results, we concluded that chitosan-based nanoparticles containing ALA were stable in stomach-like conditions and able to cross the intestinal barrier and release their antioxidant cargo. Thus, the use of α-lipoic acid in a nanoparticulate form as a dietary supplement seems to be an attractive method of administration. However, the results shown here are from in vitro experiments; thus, to

determine potentially better bioavailability than the native compound, adequately designed pharmacokinetic trials in animal models should be carried out.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12040417/s1, Video S1: Chitosan-ALA-NPs.

**Author Contributions:** Conceptualization, R.V.-D., A.S. and O.M.; Data curation, O.M. and R.V.-D.; Formal Analysis, K.Q., O.M. and R.V.-D.; Funding acquisition, A.S. and R.V.-D.; Investigation, K.Q., S.R.-G., S.J.Z.-M. and L.G.-D.; Methodology, A.G.-G., K.Q., C.L.-F. and R.V.-D.; Supervision: R.V.-D., O.M. and A.S.; Writing—original draft, K.Q. and R.V.-D.; Writing—review and editing, R.V.-D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Universidad Nacional Autónoma de México (PAPIIT IG200818 and IN209722).

**Institutional Review Board Statement:** Wistar rats were housed and used at Universidad Nacional Autónoma de México (UNAM) according to regulations of the Mexican government regarding the use of laboratory animals for research purposes (CICUAL-UNAM, NOM-062-ZOO-1999 4.2.2). Animals were sacrificed with CO2 and by cervical dislocation by trained personnel. This protocol was approved by the Institute's Research Ethics Committee (Comité de Ética en la Investigación, INB-UNAM) with register #065.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was funded by the Universidad Nacional Autónoma de México (PAPIIT IG200818 and IN209722). We also thank Yareth Silva-Sánchez, undergraduate student, who contributed to studies with everted intestine. We also thank Nydia Hernández Ríos (Unidad de Microscopia Confocal, INB-UNAM) and Alejandro de León Cuevas (LAVIS, UNAM).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

### *Article*

### **Antimicrobial Resistance of** *Salmonella enteritidis* **and** *Salmonella typhimurium* **Isolated from Laying Hens, Table Eggs, and Humans with Respect to Antimicrobial Activity of Biosynthesized Silver Nanoparticles**

**Rasha M. M. Abou Elez 1, Ibrahim Elsohaby 2,3,4,\*, Nashwa El-Gazzar 5, Hala M. N. Tolba 6, Eman N. Abdelfatah 7, Samah S. Abdellatif 7, Ahmed Atef Mesalam 8,\* and Asmaa B. M. B. Tahoun 7**

**Simple Summary:** *Salmonella enterica* are common foodborne pathogens that cause gastrointestinal signs in a wide range of unrelated host species including poultry and humans. The overuse of antibiotics as therapeutic agents and growth promoters in the poultry industry has led to the emergence of multidrug-resistant (MDR) microorganisms. Thus, there is a need to find alternatives to conventional antibiotics. Recently, the biosynthesized silver nanoparticles (AgNPs) have shown an excellent antimicrobial activity. In this study, we investigated the antibacterial, antivirulent, and antiresistant activities of the biosynthesized AgNPs on the MDR and virulent *S. enteritidis* and *S. typhimurium* isolated from laying hens, table eggs, and humans. The obtained results indicated that AgNPs have the potential to be effective antimicrobial agents against MDR *S. enteritidis* and *S. typhimurium* and could be recommended for use in laying hen farms.

**Abstract:** *Salmonella enterica* is one of the most common causes of foodborne illness worldwide. Contaminated poultry products, especially meat and eggs are the main sources of human salmonellosis. Thus, the aim of the present study was to determine prevalence, antimicrobial resistance profiles, virulence, and resistance genes of *Salmonella* Enteritidis (*S. enteritidis*) and *Salmonella* Typhimurium (*S.* Typhimurium) isolated from laying hens, table eggs, and humans, in Sharkia Governorate, Egypt. The antimicrobial activity of Biosynthesized Silver Nanoparticles (AgNPs) was also evaluated. *Salmonella* spp. were found in 19.3% of tested samples with laying hens having the highest isolation rate (33.1%). *S.* Enteritidis) (5.8%), and *S.* Typhimurium (2.8%) were the dominant serotypes. All isolates were ampicillin resistant (100%); however, none of the isolates were meropenem resistant. Multidrug-resistant (MDR) was detected in 83.8% of the isolates with a multiple antibiotic resistance index of 0.21 to 0.57. Most isolates (81.1%) had at least three virulence genes (*sopB*, *stn,* and *hilA*) and none of the isolates harbored the *pefA* gene; four resistance genes (*blaTEM*, *tetA*, *nfsA,* and *nfsB*) were detected in 56.8% of the examined isolates. The AgNPs biosynthesized by *Aspergillus niveus* exhibit an absorption peak at 420 nm with an average size of 27 nm. AgNPs had a minimum inhibitory concentration of 5 μg/mL against *S. enteritidis* and *S. typhimurium* isolates and a minimum bactericidal

**Citation:** Abou Elez, R.M.M.; Elsohaby, I.; El-Gazzar, N.; Tolba, H.M.M.; Abdelfatah, E.N.; Abdellatif, S.S.; Mesalam, A.A.; Tahoun, A.B.M.B. Antimicrobial Resistance of *Salmonella enteritidis* and *Salmonella typhimurium* Isolated from Laying Hens, Table Eggs, and Humans with Respect to Antimicrobial Activity of Biosynthesized Silver Nanoparticles. *Animals* **2021**, *11*, 3554. https:// doi.org/10.3390/ani11123554

Academic Editors: Antonio Gonzalez-Bulnes and Nesreen Hashem

Received: 1 November 2021 Accepted: 10 December 2021 Published: 14 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

concentration of 6 and 8 μg/mL against *S. enteritidis* and *S. typhimurium* isolates, respectively. The bacterial growth and gene expression of *S. enteritidis* and *S. typhimurium* isolates treated with AgNPs were gradually decreased as storage time was increased. In conclusion, this study indicates that *S. enteritidis* and *S. typhimurium* isolated from laying hens, table eggs, and humans exhibits resistance to multiple antimicrobial classes. The biosynthesized AgNPs showed potential antimicrobial activity against MDR *S. enteritidis* and *S. typhimurium* isolates. However, studies to assess the antimicrobial effectiveness of the biosynthesized AgNPs in laying hen farms are warranted.

**Keywords:** *Salmonella*; antimicrobial agents; virulence genes; resistance genes; silver nanoparticles; expression

### **1. Introduction**

*Salmonella enterica* are common foodborne pathogens, causing 93.8 million cases of gastroenteritis and 155,000 deaths worldwide and severe economic losses [1]. *Salmonella* Enteritidis (*S. enteritidis*) and *Salmonella* Typhimurium (*S. typhimurium*) are the dominant nontyphoidal serovars that cause mild gastrointestinal signs in a wide range of unrelated host species and severe infections in infants, the elderly, and immunocompromised individuals [2]. Nontyphoidal human salmonellosis is mostly associated with the ingestion of contaminated poultry meats and raw eggs [3]. However, table eggs get contaminated with *Salmonella* via horizontal transmission from the feces of infected laying hens, vertical transmission through the yolk, albumen, or eggshell membranes before oviposition and contamination of the eggshell after oviposition by infected environmental dusts [4].

Antibiotics have been extensively used in developing countries as therapeutic agents and growth promoters in laying hen farms as well as treatment for various human diseases, resulting in the emergence of antimicrobial resistance bacteria [5]. Antibiotic-resistant bacteria can be transmitted to humans either directly through the food chain or indirectly by transferring their antimicrobial resistance genes to human pathogens by mobile genetic elements associated with conjugative plasmids [6]. The presence of various virulence genes in the chromosome and plasmids of *Salmonella* plays a role in the pathogenesis of such bacteria inside the host [7]. *Salmonella* outer proteins (*sops*) and *hilA* virulence genes contribute to the invasion of host epithelial cells [8]. However, the plasmid-encoded fimbriae (*pefA*) gene is mediated through the adherence of *Salmonella* to intestinal epithelial cells [9], whereas the enterotoxin (*stn*) gene is responsible for enterotoxin production and diarrhea in the host [8]. *Salmonella* plasmid virulence (*spvs*) has relevance to *Salmonella* survival and replication inside the host [10]. Therefore, understanding the antibiotic resistance mechanisms will assist in the control and reduction in the spread of resistant bacteria. Furthermore, assessing the distribution of resistance genes in bacterial populations is an additional tool to understand the antimicrobial resistance epidemiology [11]. Thus, the development of an alternatives natural antimicrobial agent is needed.

Nanoparticles are one of the alternatives that could be used as antimicrobial agents in humans and animals [12]. Silver nanoparticles (AgNPs) are among the nanoparticles that have been used for many years in different applications including wound treatment. AgNPs physical, chemical, and biological properties depend on their size and shape [13]. AgNPs have good antimicrobial activity against bacteria, fungi, and viruses, with low cytotoxicity to mammalian cells due to their nanoscale size and various shapes [14]. The mechanism behind AgNPs's actions includes their induction of cell death through generation of reactive oxygen species [15]. Furthermore, there are different factors that make AgNPs suitable for use as antimicrobial: (1) It is simple and safe to synthesize on a large scale with low cost [16]; (2) bacterial resistance to AgNPs is extremely rare; and (3) the surface of AgNPs can be easily modified and synthesized in various shapes [17]. Several studies have reported that AgNPs have antimicrobial activity against a variety of pathogens, including Salmonella

enterica, *E. coli* O157:H7, Pseudomonas aeruginosa, Listeria monocytogenes, Klebsiella pneumoniae [18], and mastitis pathogens [19].

This study aimed to (1) determine the prevalence of *Salmonella* spp. in laying hens, table eggs, and humans, in Sharkia Governorate, Egypt; (2) detect the antimicrobial resistance profiles and virulence and resistance genes of *S. enteritidis* and *S. typhimurium* isolates; and (3) prepare and assess the antibacterial, antivirulent, and antiresistant activities of AgNPs biosynthesized by *Aspergillus niveus* (*A. niveus*) on the MDR and virulent *S. enteritidis* and *S. typhimurium* isolates.

### **2. Materials and Methods**

### *2.1. Sample Collection*

A total of 431 samples were collected from laying hen farms, egg retailers, and outpatient clinics in Sharkia Governorate, Egypt, between August 2019 and January 2020. Samples included 166 fecal swabs from laying hens, 165 table eggs, and 100 human stool samples. Aseptic techniques were strictly maintained during sample collection. Table eggs were individually packed in separate sterile plastic bags from egg retailers and brought to the laboratory. All human participants provided verbal or written consent to join the study.

### *2.2. Salmonella spp. Isolation*

*Salmonella* was isolated from eggs and fecal samples as previously described Im, et al. [20], Yang, et al. [21]. The collected eggs were disinfected with 75% alcohol, the shell was removed and the yolks and whites were mixed. A 25 mL of the sample was added to 225 mL of sterile buffered peptone water (BPW; Oxoid, Basingstoke, Hampshire, UK) and then incubated at 37 ◦C for 24 h. A 100 μL of incubated BPW suspension was selectively enriched in 10 mL of Rappaport–Vassiliadis medium (Difco, Detroit, MI, USA) and incubated at 42 ◦C for 24 h. A loopful (10 μL) of enriched Rappaport–Vassiliadis medium was plated onto xylose lysine deoxycholate agar (Difco, Detroit, MI, USA) and incubated at 37 ◦C for 24 h. The suspected *Salmonella* isolates were kept frozen at −80◦C in brain–heart infusion broth (Oxoid, Basingstoke, Hampshire, UK) supplemented with 15% glycerin (Synth®, São Paulo, Brazil). The presumptive colonies were picked up for purification on tryptone soya agar (Oxoid, Basingstoke, Hampshire, UK) and subjected to biotyping [22] and serotyping according to the Kauffmann–White scheme [23] to determine flagellar (H) and somatic (O) antigens using *Salmonella* antiserum (Denka Seiken, Tokyo, Japan).

### *2.3. Molecular Identification of Salmonella spp.*

The extracted DNA was examined by PCR targeting *invA* gene using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's guidelines [24]. Isolates identified as *Salmonella* spp. were subjected to identification of *sefA* gene (310 bp) for *S. enteritidis* [25] and *STM4495* gene (915 bp) for *S. typhimurium* [26]. PCR-confirmed *S. enteritidis* and *S.* Typhimurium isolates were screened to detect *sopB* [8], *stn* [9], *pefA* [9], *spvC* [8], and *hilA* [21] virulence genes. *S. typhimurium* ATCC 14028 and *S. enteritidis* ATCC 13076 were used as positive controls. However, *Escherichia coli ATCC 25922 was used as a negative control. The positive and negative controls were donated by the Biotechnology Unit, Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, Giza, Egypt, and were run alongside the tested isolates.*

### *2.4. Antimicrobial Susceptibility Test*

Antimicrobial resistance of *S. enteritidis* and *S. typhimurium* isolates was determined using the Kirby–Bauer disk diffusion method on Mueller–Hinton Agar (MHA) (Oxoid, Hampshire, UK), in accordance with the guidelines of the Clinical and Laboratory Standards Institute [27]. The antibiotic disks used in this study were ampicillin (AMP, 10 μg), ampicillin/sulbactam (SAM, 20 μg), amoxicillin-clavulanate (AMC, 30 μg), cefotaxime (CTX, 30 μg), imipenem (IPM, 10 μg), gentamicin (GEN, 10 μg), tetracycline (TET, 30 μg), ciprofloxacin (CIP, 5 μg), nalidixic acid (NAL, 30 μg), trimethoprim-sulfamethoxazole (SXT,

25 μg), chloramphenicol (CHL, 30 μg), azithromycin (AZM, 15 μg), nitrofurantoin (NIT, 30 μg), and meropenem (MEM, 10 μg). The zone of inhibition, measured and compared with the world standards [27], was reported as resistant (R), intermediate (I), and sensitive (S). Isolates resistant to ≥3 different antimicrobial classes were considered MDR [28]. For all isolates, the multiple antibiotic resistance (MAR) index was determined using the formula: a/b (where "a" is the number of antimicrobial agents to which an isolate was resistant and "b" is the total number of antimicrobial agents tested) following the protocol designated by Krumperman [29].

*S. enteritidis* and *S. typhimurium* isolates with phenotypic resistance to specific antimicrobial agents were selected and tested for the presence of relevant resistance genes using PCR. The targeted genes included β-lactams (*blaTEM*) [30], tetracyclines (*tetA* and *tetB*) [31,32], and nitrofurans (*nfsA* and *nfsB*) [33].

### *2.5. Biosynthesis and Characterization of AgNPs*

The AgNPs were biosynthesized from contaminated soil samples containing wastes from ceramics and photographic industries (10th of Ramadan City, Sharkia, Egypt) as described by [34]. Fungal isolate was screened to reduce the AgNo3 solution at 1 mM to AgNPs according to El-Gazzar and Rabie [35]. The available salt of AgNO3 was donated from the NanoTech Egypt (Dreamland, Egypt). The physical properties and concentrations of the biosynthesized AgNPs were investigated.

The extracted DNA of the most potent suspected fungal isolate producing AgNPs was molecularly identified at the Biology Research Unit of the Assiut University using Patho Gene-spin DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) following the manufacturer's guidelines [36]. Isolates identified as *Aspergillus* spp. were further sequenced to target the same primers (SolGent, Daejeon, South Korea). The obtained sequence was analyzed using the BLAST tool and was placed in the GenBank (accession number MT319815). The nucleotide sequence was aligned with other sequences available at the GenBank to construct a phylogenetic tree using the MegAlign of DNASTAR program package (MegAlign 5.05, DNASTAR Inc., Madison, WI, USA). The recovered fungal isolate was characterized according to DeAlba-Montero, et al. [37] using different tools such as ultraviolet (UV)–visible spectrophotometer (T80 + UV Flash Spectrophotometer, PG Instruments Ltd., Wibtoft, UK); dynamic light scattering (DLS) system; transmission electron microscope (TEM) (JEOL.JEM.1010) at an accelerating voltage of 200 Kv; Fourier transform infrared spectroscopy (FTIR, Thermo Scientific Nicolet 6700 spectrometer) in the range of 400–4000 cm−1 and a resolution of 4 cm−1; and zeta potential analyzer (ZEN 1600, Malvern, UK).

### *2.6. Antimicrobial Activity of Biosynthesized AgNPs*

The disk diffusion agar method was used to test the antimicrobial activity of biosynthesized AgNPs against *S. enteritidis* and *S. typhimurium* isolates [38]. A 10 μg of the prepared nanoparticles were paced on standard disks with a diameter of 6 mm (Padtanteb, Qods, Iran). The *S. enteritidis* and *S. typhimurium* isolates were spread on the Mueller–Hinton broth (MHB) (Merck, Darmstadt, Germany). The disks were placed on the agar plate and a sterile blank disk was used as control; then, the inhibition zone was measured after a 24 h incubation at 37 ◦C.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of biosynthesized AgNPs were determined using tube dilution method as previously described Krishnan, et al. [39]. Bacterial inoculums were adjusted to match a 0.5 McFarland turbidity standard. A 100 μL of AgNPs with different concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg/mL) from each speed (4000, 8000, and 14,000 rpm) and a 100 μL from the tested organism were applied to 5 mL MHB and incubated at 37 ◦C with shaking for 24 h. Broth media with AgNPs inoculum was used as a positive control. The bacterial growth was monitored by measuring the mean OD at 600 nm.

The MBC was determined by coating the bacterial inoculum with different nanoparticle suspension concentrations into the MHA plate and then incubated at 37 ◦C for 24 h. The MBC value was defined as the lowest concentration, with no visible growths on the MHA plate.

### *2.7. Virulence and Resistance Genes Expression*

### 2.7.1. Bacterial Counting

The experiment used 2 MDR *Salmonella* isolates (*S. enteritidis* and *S. typhimurium* isolated during our study and were positive for most virulence and resistance genes). The bacterial inoculums were adjusted to 1.5 × 108 Colony Forming Unite (CFU)/mL. The surface plating method was used for the bacterial count on an agar plate [40]. Bacterial inoculums were inoculated with a final concentration of 5 μg/mL followed by constant stirring to obtain a uniform colloidal nanoparticle suspension. Nanoparticle-free medium was used as a positive control, whereas the bacteria-free medium was used as a negative control. Each inoculated suspension was incubated at 37 ◦C for 0, 12, 24, 36, and 48 h. Bacterial growth inhibition was determined using the surface plating method by counting the number of CFUs on the plates. Bacteria and nanoparticle mixtures were prepared at different cultivation times. The amplification of the 16S rRNA gene was used to molecularly confirm *Salmonella* colonies. Bacterial cells were counted in triplicate and then the mean values and standard deviations were calculated.

### 2.7.2. Quantitative Reverse Transcription PCR Analysis of Genes Expression

At each sampling time, 1 volume of the harvested bacterial culture was added to 1 volume of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) following the manufacturer's instructions. RNA isolation was implemented using QIAamp RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's guidelines. SYBR Green I-based real-time PCR with the specific primers for *sopB*, *stn,* and *hilA* virulence genes and *blaTEM*, *tetA,* and *nfsA* resistance genes (which are all present in the used 2 *Salmonella* isolates) was performed and the 16S rRNA gene was used as a housekeeping gene [21]. The primers were used in a 25 μL reaction containing 12.5 μL of 2× QuantiTect SYBR Green PCR Master Mix (QIAGEN), 0.25 μL of RevertAid Reverse Transcriptase (200 U/μL) (Thermo Fisher, Waltham, MA, USA), 0.5 μL of each primer (20 pmoL concentration), 8.25 μL of nuclease-free water, and 3 μL of RNA template. The reaction was performed in a Stratagene MX3005P real-time PCR machine where the amplification curves and cycle threshold (Ct) values were determined by Stratagene MX3005P software. The comparative Ct method was used to estimate the variations in RNA gene expression of the different samples by comparing Ct of each sample with that of the positive control. The ΔΔCt method was performed, according to Yuan, et al. [41].

### *2.8. Statistical Analysis*

The R software (R Core Team, 2019; version 3.5.3) was used for the descriptive and statistical analysis. A heatmap was constructed based on the virulence and resistance genes and the antimicrobial susceptibility results using the R package "Complex heatmap" [42]. Nonmetric multidimensional scaling (nMDS) [43] was performed using the "metaMDS" function in "vegan" package to compare the dissimilarity of antimicrobial resistance profiles, using Bray–Curtis distance among isolates across all *Salmonella* spp. and within each species. The "corrplot" function in "corrplot" package was used to assess the correlation between the antimicrobial resistance and the presence of virulence and resistance genes. One-way analysis of variance was used to determine the significant difference between the bacterial counts and the fold change values of the gene's expression at each storage time. Multiple comparisons between the means were assessed by the Tukey's honestly significant difference test. *p* < 0.05 was considered statistically significant.

### **3. Results**

### *3.1. Salmonella spp. Isolation and Identification*

*Salmonella* spp. were identified in 83 of 431 examined samples (19.3%) and were mostly identified in samples from laying hens (33.1%) followed by humans (22%) and table eggs (3.6%) (Table 1). The most dominant *Salmonella* serotypes were *S. enteritidis* (5.8%) and *S. typhimurium* (2.8%), followed by *S. kentucky* (1.9%), *S. virchow* (1.6%), *S. tamale*, *S. inganda*, *S. wingrove*, *S. bargny* (1.2%, each), *S.* Anatum (0.9%), *S. tsavie*, *S. larochelle* (0.7%, each), and *S. apeyeme* (0.2%). On the other hand, only *S. apeyeme* was isolated from human samples (1 isolate), whereas *S. anatum*, *S. tsavie*, and *S. larochelle* were not identified (Table 1).

**Table 1.** Serotypes and pathotypes of *Salmonella* spp. isolated from laying hens, table eggs and humans.

### *3.2. Antimicrobial Susceptibility Test*

The antimicrobial susceptibility profiles of the 25 *S. enteritidis* and 12 *S. typhimurium* isolates against 14 antimicrobial agents are presented in Table 2. All *S. enteritidis* and *S. typhimurium* isolates were resistant to AMP (100%) and sensitive to MEM (100%). In addition, *S. enteritidis* and *S. typhimurium* isolates exhibited high rates of resistance to tetracycline, NIT, and amoxicillin (Figure 1). The MDR was observed in 28 isolates (75.7%) with a MAR index ranging from 0.21 to 0.57.

**Table 2.** The antimicrobial resistance profile of *S. enteritidis* (*n* = 25) and *S. typhimurium* (*n* = 12) isolated from laying hens, table eggs, and humans.

R = resistant, I = intermediate resistance, S = sensitive.

**Figure 1.** Heat map representation of the virulence and resistance genes and antimicrobial resistance profiles of *S. enteritidis* (*n* = 25) and *S. typhimurium* (*n* = 12) isolated from laying hens, table eggs, and humans.

The nMDS plot (Figure 2) shows that the antimicrobial profiles of *S. enteritidis* and *S. typhimurium* isolated from chickens, eggs, and humans' samples were overlapped. The plot shows no evidence for clustering by isolate source.

**Figure 2.** Non-metric multidimensional scaling ordination (NMDS) of antimicrobial-resistant of *S. enteritidis* and *S. typhimurium* isolated from laying hens, table eggs and humans.

### *3.3. Virulence and Resistance Genes*

The presence of virulence and resistance-associated genes in *S. enteritidis* and *S. typhimurium* isolates is presented in Table 3. The *SopB* gene was identified in all *Salmonella* spp. isolates, whereas the *pefA* gene was not detected in any isolate. The *stn* virulenceassociated gene was identified in all *S. enteritidis* and 75% of *S. typhimurium* isolates. The *hilA* gene was mostly identified in 94.6% of *Salmonella* isolates; however, the *SpvC* virulence gene was only detected in 5.4% of *S.* Typhimurium isolates. All *Salmonella* isolates (100%) harbored only 1 virulence gene and 34 isolates (91.9%) were positive for >1 virulence gene (Figure 1).

Five resistance genes (*blaTEM*, *tetA*, *tetB*, *nfsA* and *nfsB*) were also identified in *S. enteritidis* and *S. typhimurium* isolates (Table 3). The *blaTEM* gene was identified in all isolates and most isolates harbored *nfsA* (94.6%) and *nfsB* and *tetA* (83.8%, each) resistance genes. However, the *tetB* resistance gene was only recognized in two *S. enteritidis* isolates. Notably, five resistance profiles were exhibited in the study and most isolates (56.8%) were related to a profile carrying four resistance genes (*blaTEM*, *tetA*, *nfsA* and *nfsB*) (Figure 1).

Significant correlations between resistance genes and antimicrobial agents are presented in Figure 3. There were significant positive correlations between resistance genes and their corresponding antimicrobial agents in most cases. The analysis also showed a significant negative correlation between resistance genes and antimicrobial agents other than the corresponding ones.

**Figure 3.** Correlation matrix showing the correlation between resistance phenotypes and genotypes among the examined *S. enteritidis* and *S. typhimurium* isolates recovered from laying hens, table eggs, and humans. The blue colour indicates a positive correlation and red shows a negative correlation. The asterisk (\*) indicates significant at *p* < 0.001.

### *3.4. Characterization of Biosynthesized AgNPs*

AgNPs were synthesized from *Aspergillus niveus* (*A. niveus*; MT319815), molecularly confirmed in the study targeting the 28S rRNA region (Figure 4A). AgNPs were oval, cubic, rod shaped, and well distributed without agglomeration at 6.49 nm using TEM microscopy (Figure 4B). The prepared AgNPs were characterized by the absorption peak of AgNPs at 420 nm using the UV–visible spectrum and an average particle size at 27 nm using DLS (Figure 4C). FTIR spectra of AgNPs confirmed the presence of various functional groups at 3451.81, 2081.40, 1634.19, 1384.39, 1117.76, and 614.02 cm−1. The peaks at 3451.81, 2081.40, 1634.19, and 614.02 cm−1 correspond to carbonyl residues, alcohol, nitrile, acid chloride, and alkene band C–C stretch in ring of CH3, stretch of alkyl halides and peptide bonds of proteins responsible for the synthesis of the AgNPs (Figure 4D). The bands at 3451.81, 1634.19, and 1384.39 cm−1 correspond to the binding vibrations of amide I and amide II of protein and hydroxyl O–H stretch of phenols with amine N–H stretchings. In addition, the band at 1117.76 cm−1 refers to one mononuclear aromatic. Dynamic light scattering analysis showed that the average particle size of the prepared AgNPs was at 27 nm (Figure 4E). The stability degree (Zeta Potential) of AgNPs showed a negative charge at −30.4 mv (Figure 4F).

### *3.5. Antimicrobial Activity of Biosynthesized AgNPs*

The disk diffusion method was used to evaluate the antimicrobial activity of *S. enteritidis* and *S. typhimurium* isolates treated with AgNPs. The diameter of inhibition zones of AgNP (10 μg)-treated *S.* enteritidis and *S. typhimurium* isolates were 24 mm and 20 mm, respectively. The MIC for *Salmonella* treated with different concentrations (10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 μg/mL) in this study was 5 μg/mL upon using the dilution method to *S. enteritidis* and *S. typhimurium* isolates, whereas MBC value was 6 μg/mL to *S. enteritidis* isolates and 8 μg/mL to *S. typhimurium* isolates.

The selected MBC value for each strain was evaluated by assessing the bacterial growth (CFU/mL) at time intervals 12, 24, 36, and 48 h (Figure 5). The growth of *S. enteritidis* and *S*. *typhimurium* significantly (*p* < 0.05) declined at 12 h and reached complete inhibition at 48 h. In parallel, the control plates showed a significant increase in growth.

**Figure 4.** Characterization of AgNPs. (**A**) Phylogenetic analysis of the *Aspergillus niveus* (MT319815) used in the biosynthesis of AgNPs; (**B**) TEM micrographs and size distribution for silver (scale bar: 100 nm); (**C**) UV–Visible spectrum of AgNPs; (**D**) Fourier transform infrared spectrum showing the functional groups on the surface of AgNPs; (**E**) Dynamic light scattering analysis showing the highest peak at 27 nm and (**F**) Zeta-potential of AgNPs.

**Figure 5.** Bacterial growth of multidrug-resistant *S. enteritidis* and *S. typhimurium* treated with AgNPs. Bacterial counts were recorded at 0, 12, 24, 36, and 48 h post-treatment.

### *3.6. Antivirulent and Antiresistant Activity of Biosynthesized AgNPs*

The expression patterns of virulence and resistance genes of *S. enteritidis* and *S. typhimurium* isolates treated with AgNPs at time intervals 6, 12, 24, and 36 h are presented in Table 4. The expression levels of virulence and resistance genes significantly decreased by time. Despite the loss of expression of the virulence genes at 36 h, the resistance genes showed instant expression.

**Table 4.** Virulence and resistance genes expression of multidrug-resistant *S. enteritidis* and *S. typhimurium* treated with AgNPs.

### **4. Discussion**

Chicken and eggs are considered one of the main reservoirs for zoonotic pathogens [44]. In this study, 12 different *Salmonella* serovars were identified in the examined samples with *Salmonella* spp. prevalence of 19.3%, and most of them were identified in laying hen samples (33.1%). The observed prevalence in laying hens was comparable to the 32.0% reported recently in Egypt [45] and the 33.3% in Colombia [46], but higher than the 1.02% and 10.4% reported in Spain [47] and Egypt [48], respectively. However, the prevalence of *Salmonella* spp. infection in laying hens was lower than the 59.3% that was reported previously in Korea [20]. Furthermore, the prevalence of *Salmonella* spp. infection in human samples (22%) was higher than the 4% that was reported previously in the same governorate [49]. In this study, the isolation rate of *Salmonella* spp. from egg content was low (3.6%) and lower than the 5.2% reported by Im, Jeong, Kwon, Jeong, Kang, and Lee [20] and the absence of *Salmonella* in egg content reported by Zubair, et al. [50]. The low incidence of *Salmonella* in egg content was caused by the egg's complex system of membrane barriers and the albumen antibacterial effect [51]. Furthermore, *Salmonella* in egg contents may be attributed to contamination of eggshell by *Salmonella* infected poultry feces, which may penetrate the interior of eggs and grow during storage [52].

*S. enteritidis* and *S. typhimurium* were the most commonly isolated serotypes in this study, which was consistent with previous reports from Egypt [45] and Brazil [53]. The *S. enteritidis* and *S. typhimurium* isolates used in this study showed a high rate of antimicrobial resistance (100%) to at least one antimicrobial agent. Antimicrobial agents (AMP, TET, and NIT) were not effective against *Salmonella* isolates in this study. Our findings were comparable to those reported previously in South India [54] and Ghana [55]. The high rate of resistance reported in this study could be attributed to a number of factors, including the overuse of antimicrobial agents (over-the-counter antibiotics without a prescription) and the inappropriate use of antimicrobial agents as a growth promoter in animals, which could lead to the emergence of resistant bacteria in both animals and humans through direct contact or through the food chain [56]. All *Salmonella* isolates in this study were sensitive to SAM and MEM, which was consistent with previous Egyptian reports [57,58], and was accredited to the limited use of these antimicrobial agents in commercial chicken farms.

Previously, MDR from various sources has been identified in *Salmonella* [57,59], making these antimicrobial agents ineffective in humans and poultry [60]. In the current study, 75.7% of *Salmonella* isolates displayed MDR, which is higher than the prevalence of MDR *Salmonella* isolated from chicken in the USA [61] and China [62,63]. Despite this, the prevalence of MDR *Salmonella* in this study was lower than the 100% previously reported in chicken in Egypt [57] and 92.9% in North India [59]. The resistant isolates were not evenly distributed in laying hens, eggs, and humans. However, these isolates were overlapped, reflecting the high public health impact of MDR isolates and the need for new antimicrobial agents to treat human salmonellosis [64].

The presence of both virulence and antimicrobial resistance genes affects the bacteria' pathogenicity [8]. The emergence of MDR strains of *Salmonella* is primarily due to genetic factors that enhance their survival by retaining drug resistance genes [65]. Of the confirmed *S. enteritidis* and *S. typhimurium* isolates, 81.1% harbored three virulence genes (*sopB*, *stn,* and *hilA*), which was similar to the previous study in Malaysia [66]. None of the isolates harbored *pefA* gene in our study, which was similar to a previous study in Egypt [45]. In contrast to Thung, Radu, Mahyudin, Rukayadi, Zakaria, Mazlan, Tan, Lee, Yeoh, and Chin [66], who reported the absence of *spvC* gene *Salmonella* isolates, the *spvC* was identified in 5.4% of *S. enteritidis* and *S. typhimurium* isolates.

In the present study, *Salmonella* harbors a range of antibiotic resistance genes located on mobile genetic elements that distribute resistance characteristics to other serovars and other different bacteria [59]. All isolates had *blaTEM* resistance genes and most of them had *tetA*, *nfsA,* and *nfsB* resistance genes, which was similar to *Salmonella* isolates from chickens in Egypt [57] and North India [59]. The *TetB* was not identified in previous studies, but *tetA* was the most common, highlighting its resistance role to tetracyclines [59,67].

There is currently a need for more research to find novel materials that inhibit antimicrobial-resistant variants [68]. Therefore, the development of natural antimicrobial agents such as AgNPs may be an alternative way to overcome MDR bacteria. In our study, AgNPs were biosynthesized from molecularly confirmed *A. nivus*. in line with the previous study by Elgazzar and Ismail [69], the peak of AgNPs using UV–visible spectra was witnessed at 420 nm. The small particle size of AgNPs (27 nm) with a single peak indicates the suitable quality of the biosynthesized nanoparticles and ideal inhibitory effects on bacterial growth [70]. The small particle size allows AgNPs to bind to the cell wall and easily penetrate the bacteria cell, enhancing their antimicrobial activity against the bacteria owing to their larger surface area and greater interaction [71]. The TEM microscopy indicated that biosynthesized AgNPs were well dispersed in the solution without agglomeration, explaining that fungal filtrates contain various biomolecules used to cap and stabilize the agglomeration of AgNPs [35]. FTIR measurements of biosynthesized AgNPs have verified different functional groups of biomolecules and capped proteins, excreted by the fungus itself, encapsulated nanoparticles, and increased stability; associated proteins may help to mineralize precursor salts [69]. The higher negative charge of the AgNPs as measured by the zeta potential confirms the synthesized particles' repulsion, resulting in stability and monodispersity of the synthesized AgNPs solution [72].

In this study, a clear zone was found to exist around the AgNP disk, suggesting that the biosynthesized AgNPs possessed a potent antibacterial effect against the growth of MDR bacteria, causing a reduction in bacterial number [73,74]. To evaluate the effect of AgNPs against *Salmonella*, MIC and MBC values were examined in our study, showing the extent of variation with Loo, et al. [74], who reported that the MIC value of *S. enteritidis* and *S. typhimurium* was 3.9 mg/mL (each) and the MBC values for *S. enteritidis* and *S. typhimurium* were 3.9 mg/mL and 7.8 mg/mL, respectively.

The bacterial growth inhibition of AgNPs occurs because AgNPs interfere with sulfur found in biomolecules on the bacterial membrane, attacking the bacterial genome and resulting in bacterial death [75]. In this study, a significant decrease was found in the number of *Salmonella* isolates after different storage time and the growth was completely absent after 48 h of incubation; however, Tanhaeian and Ahmadi [76] reported a decline in the bacterial growth treated with 25 μg/mL and 50 μg/mL of AgNP after 10 h and the growth level dropped to almost zero.

Besides exploring the effect of the biosynthesized AgNPs on the growth inhibition of *Salmonella*, we investigated their impact on the expression of genes that are essential to the virulence and resistance of the pathogen. The results emphasized that biosynthesized AgNPs at a concentration of 5 μg significantly reduce gene expression, as previously reported [76]. The adverse effect of the biosynthesized AgNPs on virulence and resistance genes expression contributes to effective preventative and potent antimicrobial drugs against bacterial infection [76]. AgNPs are broad-spectrum antimicrobial agents that have the same effect on all Gram-negative bacteria strains. However, the concentration of the used AgNPs and bacteria class, nanoparticle size, synthesis method, and physical characteristics of nanoparticles are key factors that can affect the results across studies.

### **5. Conclusions**

In the present study, *S. enteritidis* and *S. typhimurium* were the most common *Salmonella* serovars isolated from laying hens, table eggs, and humans in Egypt. Moreover, most of the recovered *Salmonella* isolates exhibited MDR, which poses a possible risk to consumers in Egypt. The findings of this study support that AgNPs have the potential to be effective antimicrobial agents against MDR *S. enteritidis* and *S. typhimurium* and could be recommended for use in laying hen farms. However, further studies are required to develop and design a safe AgNPs antimicrobial for laying hen farms.

**Author Contributions:** Conceptualization, R.M.M.A.E.; I.E.; N.E.-G.; H.M.N.T.; E.N.A.; A.A.M.; S.S.A., and A.B.M.B.T.; methodology, R.M.M.A.E.; N.E.-G.; H.M.N.T.; E.N.A.; A.A.M.; S.S.A., and A.B.M.B.T.; software, R.M.M.A.E.; I.E., and N.E.-G.; validation, R.M.M.A.E.; N.E.-G.; H.M.N.T.; E.N.A.; A.A.M.; S.S.A., and A.B.M.B.T.; formal analysis, I.E.; investigation, R.M.M.A.E. and A.B.M.B.T.; resources, N.E.-G.; H.M.N.T.; E.N.A.; A.A.M., and S.S.A.; data curation, R.M.M.A.E.; N.E.-G.; A.A.M., and A.B.M.B.T.; writing—original draft preparation, R.M.M.A.E.; I.E.; N.E.-G., and A.B.M.B.T.; writing—review and editing, R.M.M.A.E.; I.E.; N.E.-G.; H.M.N.T.; E.N.A.; A.A.M.; S.S.A., and A.B.M.B.T.; visualization, R.M.M.A.E.; I.E.; N.E.-G., and A.B.M.B.T.; supervision, R.M.M.A.E.; project administration, R.M.M.A.E. and A.B.M.B.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** No specific funding was received.

**Institutional Review Board Statement:** This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Zagazig University (Ref. No.: ZU-IACUC/2/F/7/2020).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank the participants (laying hens farms and patients) who agreed to use their samples in this study. Authors would also thank the Egyptian Knowledge Bank (EKB) for editing and grammar correction of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

### *Article* **Anisotropic Silver Nanoparticles Gel Exhibits Antibacterial Action and Reduced Scar Formation on Wounds Contaminated with Methicillin-Resistant** *Staphylococcus pseudintermedius* **(MRSP) in a Mice Model**

**Saengrawee Thammawithan 1,2, Oranee Srichaiyapol 1, Pawinee Siritongsuk 1,2, Sakda Daduang 2,3, Sompong Klaynongsruang 1,2, Nuvee Prapasarakul 4 and Rina Patramanon 1,2,\***

**Simple Summary:** Wound infection in animals with antimicrobial resistant bacteria, especially *Staphylococcus pseudintermedius*, plays an important role in the delay of wound healing. In this work, the antimicrobial and wound healing activities of gels containing anisotropic AgNPs were evaluated on wounds contaminated with Methicillin-resistant *Staphylococcus pseudintermedius* in a mice model. The results show that anisotropic AgNPs gel is effective in eliminating bacteria and preventing pus formation. Furthermore, anisotropic AgNPs gel exhibits improved collagen alignment that supports scar disappearance.

**Abstract:** *Staphylococcus pseudintermedius* (*S. pseudintermedius*) infected wounds can cause seriously delayed wound healing processes in animals. Antimicrobial agents that have antimicrobial and wound healing efficacy have become an essential tool for overcoming this problem. In our previous study, anisotropic AgNPs have been reported to have antimicrobial efficiency against animal and human pathogens, and could be suitable as antimicrobial agents for infected wounds. Here, antimicrobial and wound healing activities of anisotropic AgNPs gels were assessed *in vivo*. BALB/cAJcl mice wounds were infected by Methicillin-resistant *Staphylococcus pseudintermedius* (MRSP). Then, antibacterial and wound healing activities were evaluated by bacterial cell count, wound contraction, digital capture, and histology. The results show that anisotropic AgNPs gels could eliminate all bacterial cell infected wounds within 7 days, the same as povidone iodine. Wound healing activity was evaluated by wound contraction (%). The results showed 100% wound contraction in groups treated with anisotropic AgNPs gels within 14 days that was not significantly different from povidone iodine and control gel without AgNPs. However, the digital capture of wounds on day 4 showed that anisotropic AgNPs gel prevented pus formation and reduced scar appearance within 21 days. The histology results exhibit improved collagen fiber alignment that supports scar disappearance. In conclusion, these results indicate that anisotropic AgNPs gels are suitable for treating infected wounds. The gel is effective in eliminating bacteria that supports the natural process of wound repair and also causes reduced scar formation.

**Keywords:** *Staphylococcus pseudintermedius*; wound infection; antimicrobial resistance; alternative antimicrobial agent; nanotechnology; silver nanoparticles; scar reduction

**Citation:** Thammawithan, S.; Srichaiyapol, O.; Siritongsuk, P.; Daduang, S.; Klaynongsruang, S.; Prapasarakul, N.; Patramanon, R. Anisotropic Silver Nanoparticles Gel Exhibits Antibacterial Action and Reduced Scar Formation on Wounds Contaminated with Methicillin-Resistant *Staphylococcus pseudintermedius* (MRSP) in a Mice Model. *Animals* **2021**, *11*, 3412. https://doi.org/10.3390/ani11123412

Academic Editors: Antonio Gonzalez -Bulnes and Nesreen Hashem

Received: 11 October 2021 Accepted: 27 November 2021 Published: 30 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

### **1. Introduction**

Bacterial infections can easily occur when animals have a wound that breaks the skin. Contamination at the wound site leads to a delay of wound healing and permits bacteria to spread to other organs. In animals in particular, wounds caused by bites are a common way that animals can get a bacterial infection, because their teeth are covered in bacteria [1]. Therefore, if a wound is caused by bites, they are usually more serious than they look and can easily become infected. Wound infections in animals are usually caused by opportunistic pathogens that are the normal microflora of the skin [2]. Similarly to *Staphylococcus aureus*, which is among normal flora on human skin [3], *S. pseudintermedius* is harmless in healthy individuals, but it is an opportunistic pathogen if an animal gets injured or sick [4]. These bacteria are one of the most common pathogens causing wound infection in animals [5].

*S. pseudintermedius* is a gram-positive cocci present in the skin and mucosa of animals [6]. Since it is an opportunistic pathogen in most animal species, it is an important pathogen in veterinary medicine. *S. pseudintermedius* has been isolated as a normal inhabitant of the skin, mucosa, nares, mouth, pharynx, forehead, groin and anus of healthy dogs, cats, horses, etc. [7,8]. In fact, 84.7% of all *S. pseudintermedius* isolates originate from canines [5]. *S. pseudintermedius* is a major cause of skin and ear infections, infections of other body tissues, cavities and wound infections in dogs and cats [6,9,10], and has become a pathogenic bacterium of veterinary concern, since a noted property of *staphylococci* is their ability to become resistant to antimicrobials [11,12]. It has been reported that up to 97.8% of Methicillin-resistant *S. pseudintermedius* (MRSP) isolates exhibit multidrug resistance (MDR) to three or more antibiotics routinely used in veterinary medicine [13,14]. Moreover, the bacteria is a zoonotic pathogen that can transmit to and infect humans [15,16]. Therefore, alternative antimicrobial agents to treat MRSP infected wounds are urgently needed in cases of wound infection in animals.

Silver nanoparticles have been used as a topical agent in various formulations for several hundred years; they have been used in applications as diverse as photographics, conductive/antistatic composites, catalysts, biocides and as a wound treatment. The physical, chemical and biological properties of AgNPs depend on their size and shape [17], and in addition, stabilizers are also important to these properties [18,19]. Due to their nanoscale size and the various shapes of AgNPs, they exhibit good antimicrobial action against bacteria, fungi and viruses with low cytotoxicity to mammalian cells [20]. Furthermore, the wound healing activity of AgNPs has been reported in many research studies. AgNPs promote wound healing through anti-inflammatory properties, as indicated by reduced cytokine release, decreased lymphocyte and mast cell infiltration and inhibition of matrix metalloproteinase 2 and 9 [21,22]. In addition to good antimicrobial activity and wound healing properties, AgNPs are also suitable for application as antimicrobial and wound healing agents owing to: (1) Bacterial resistance to AgNPs is extremely rare, due to the presence of multiple bactericidal mechanisms [23]; (2) The fact that the surface of AgNPs can be easily modified [24]; (3) It is easy to synthesize using large scale, inexpensive, safe and reliable methods, including chemical, physical and biological methods [25]; (4) It is easy to synthesize in different shapes (spheres, rods, hexagons, triangles, tubes, plates, cubes) [26].

Owing to AgNPs having various sizes and shapes, they can be divided into two types following their shape; silver nanospheres are spherical in shape, whereas anisotropic AgNPs are non-spherical shapes of AgNPs, such as truncated triangle, hexagon, and rod. Anisotropic AgNPs show various physical, chemical, and biological properties, such as color of colloid solution, conductivity, antimicrobial action and cytotoxicity [27]. Due to their outstanding optical properties and electrical conductivity, anisotropic AgNPs are usually used for catalysts, sensors and imaging [28,29]. However, previous reports show anisotropic AgNPs also have a good antimicrobial effect [30,31]. Moreover, many reports display that the antimicrobial activity of anisotropic AgNPs as being higher than silver nanospheres [32]. Anisotropic AgNPs, such as truncated triangular, display the strongest

antibacterial action, compared with spheres [33]. Another report has shown that silver nanoplatelets with triangular or circular shape are more toxic towards *S. aureus* than spherical and rod shapes [34]. In our previous study, anisotropic AgNPs, an appropriate mixture of nanospheres and nanoplates (i.e., circular disk, truncated triangle and hexagon), were synthesized via transformation of silver nanospheres with an optimum concentration of hydrogen peroxide. The anisotropic AgNPs have been presented with antimicrobial efficiency against animal and human pathogens with low cytoxicity to human cell lines [35]. Moreover, we have developed an antimicrobial gel containing AgNPs as an easy way to use an anisotropic AgNP-based antimicrobial formulation for topical use, such as for infected wounds. The gel can inhibit the growth of the bacterial pathogens and shows long-lasting protection against bacteria, compared with povidone iodine. Hence, in this study we evaluated antimicrobial and wound healing activities of gel containing anisotropic AgNPs toward wounds contaminated with MRSP in a mice model to assess the antimicrobial and wound healing efficacy of anisotropic AgNPs gel for alternative applications on infected wounds in animals as well as to expand their application to humans.

### **2. Materials and Methods**

### *2.1. Animals and Animal Care*

The adult female BALB/cAJcl mice of 6 weeks old (17 to 25 g) that were used in this study were supplied by Nomura Siam International Co., Ltd., Bangkok Thailand. Animals were housed in specially designed cages with a size of 26.6 × 42.5 × 21 cm (6 mice per cage), in a room with a controlled temperature (23 ± 2 ◦C), 30–60% RH, and 12 h light/dark cycle, with specific pathogenic-free conditions, at the Northeast Laboratory Animal Center, Khon Kaen University. The experimental protocol was approved by the animal ethics committee of Khon Kaen University, Khon Kaen, Thailand (Animal Ethics KKU 3/61).

### *2.2. Bacterial Strain and Culture Media*

MRSP MIC 411 (Exudate from dog wound) were kindly provided by the Department of Microbiology, Faculty of Veterinary Science, Chulalongkorn University. The bacteria were identified by the Vitek 2 Compact System (bioMérieux, Marcy l'Etoile, France). The oxacillin-resistant isolates (Minimum inhibitory concentration (MIC) ≥4 μg/mL) further underwent mecA gene existence test using the PCR method [36]. The bacteria was streaked on Mueller-Hinton agar (MHA) and incubated at 37 ◦C for 24 h. A colony was selected and inoculated with 5 mL of Mueller-Hinton broth (MHB, HiMedia Laboratories Pvt. Ltd., Bengaluru, India) at 37 ◦C overnight. Then, bacteria was sub-cultured in 5 mL of the same medium at 37 ◦C in a 180-rpm shaker-incubator for 3 h to yield a mid-logarithmic growth phase culture.

### *2.3. Anisotropic AgNPs Gel Preparation*

Tannic acid-stabilized anisotropic AgNPs were provided by our collaborator Prime Nanotechnology Co., Ltd. (Bangkok, Thailand) with a stock concentration of 100 mg/L. Anisotropic AgNPs were synthesized by transformation of silver nanospheres with an optimum concentration of hydrogen peroxide. For anisotropic AgNPs solution (a stock concentration of 100 mg/L) preparation, anisotropic AgNPs were prepared in sterile deionized water. In order to use anisotropic AgNPs in the form of a gel, poly(acrylic acid) (Sigma-Aldrich, St. Louis, MO, USA) was used to prepare the gel. Poly(acrylic acid) was chosen due its ease of preparation. Additionally, they have many advantages, such as good biocompatibility, keeping the wound moist and the ability to absorb many times their weight in water [37–39]. Briefly, 0.2 g poly(acrylic acid) was dissolved in 50 mL of sterile deionized water, followed by the addition 1 mL of glycerol (Sigma-Aldrich, St. Louis, MO, USA). After that, anisotropic AgNPs were added into the solution with final anisotropic AgNPs concentrations of 40 μg/mL (The minimum inhibitory concentration of anisotropic AgNPs against bacteria). Then, 0.2 g of triethanolamine (Sigma-Aldrich, St. Louis, MO, USA) was added to adjust pH and allow poly(acrylic acid) to form a gel. In

the final step, sterile deionized water was added into the gel to make 100 g gel, and it was stirred well. The anisotropic AgNPs gel was stored at room temperature (27–30 ◦C) until use. The antimicrobial activity of anisotropic AgNPs gel was evaluated by diffusion test before application to mice. Briefly, the bacteria at an inoculum of 1 × 107 CFU/mL was swabbed on an MHA plate. A hole with a diameter of 6 mm was punched aseptically with a sterile cork borer. Then, 0.04 mg/g of anisotropic AgNPs gel was filled into the wells at 0.2 g in triplicate and incubated at 37 ◦C for 24 h. Povidone iodine (Leopard medical brand Co., Ltd., Nakorn Pathom, Thailand) was used as the positive control, and the gel without AgNPs as the negative control. After 24 h of incubation, the inhibition zone was measured with the millimeter (mm) scale.

### *2.4. Anesthesia and Wounding*

All mice were anesthetized with intraperitoneal injection of a ketamine hydrochloride (PubChem, Bethesda, MD, USA) (90 mg/kg) and xylazine (Bimeda, Cambridge, ON, Canada) (10 mg/kg) saline cocktail [40]. The skin areas of the backs of the mice were cleaned with 70% ethanol (Siribuncha Co., Ltd., Nonthaburi, Thailand), povidone iodine (Leopard Medical Brand Co., Ltd., Nakhon Pathom, Thailand) and then 70% ethanol before wound creation. Wounds (8 mm in diameter) were made on the dorsal midline using sterile tissue scissors with curved blades. *S. pseudintermedius* MIC 411 were prepared to 107–108 CFU/mL in 5 mL of sterile Phosphate-Buffered Saline (PBS). Fifty microliters of the bacterial suspension were added to each wound bed immediately after wound surgery. After 2 days of wound creation, infected mice were divided into 3 groups (*n* = 6) including negative control (gel without AgNPs), positive control (povidone iodine; Leopard Medical Brand Co., Ltd.), and test group (anisotropic AgNPs gel). The mice had one agent applied once a day, every day for 14 days.

### *2.5. Measurement of Wound Infection and Wound Size*

Wound infection was performed using the serial dilution plate count method of bacterial load at the wound site on days 0, 4, 7, 10 and 14 before application of antimicrobial agents. Briefly, the wound sites were swabbed by cotton swab and kept in PBS buffer pH 7.4. Each sample solution was brought to 50 μL for a serial ten-fold dilution plate count (10−1–10−8 conc.) with sterile PBS buffer in triplicate. Then, 10 μL of each dilution was dropped on MHA and incubated at 37 ◦C overnight to count the bacterial colonies formed. In addition, all wounds were digitally photographed on days 1, 4, 7, 10, 14 and 21 to assess wound healing by the naked eye. Moreover, wound size was determined in diameter by using a vernier caliper on days 0, 4, 7, 10 and 14. Determinations were performed for wound area (mm2) using length and width measurements. Wound contraction was calculated using the following formula:

$$\text{Wound contraction } (\%) = \frac{A\_0 - A\_t}{A\_0} \times 100\tag{1}$$

where *A*0 is the initial wound area and *At* is the area of the wound at the time of image capture [41].

### *2.6. Histological Analysis*

The animals were euthanized via CO2 inhalation on days 14 or 21, followed by immediately taking tissue from the wound area. The tissues of 0.5 × 0.5 mm2 were fixed in 10% neutral buffered formalin (NBF) (RCI Labscan Ltd., Bangkok, Thailand) for 24 h and then embedded in paraffin. Sections were cut at a thickness of 5 μm and stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO, USA). Evaluations were made under a light microscope.

### *2.7. Statistical Analysis*

The bacteria load in the wound area and wound contraction data were expressed as the mean ± standard deviation. Analysis of variance with Tukey's test was used for multiple comparisons. A *p*-value of less than 0.05 was considered significant.

### **3. Results**

### *3.1. Antimicrobial Test of Anisotropic AgNPs Gel*

The characteristics of anisotropic AgNPs gel are shown in Figure 1. Control gel (without AgNPs) was colorless, while the gel contained anisotropic AgNPs was dark orange. Anisotropic AgNPs gel remained dark orange and was still stable after storage at room temperature after 1 year (Figure 1c). To evaluate antimicrobial activity of anisotropic AgNPs gel before in vivo testing, an antimicrobial screening test was performed by the diffusion method. As shown in Figure 2, anisotropic AgNPs gel presented an inhibition zone that indicated antimicrobial activity toward the bacteria. The antimicrobial activity of anisotropic AgNPs gel was tested after storage at room temperature for a year. The testing exhibited an inhibition zone at both 24 h and 1 year of storage. The average size of the inhibition zone had no significant differences at 16.37 ± 1.09 mm and 15.67 ± 0.98 mm when the gel was stored for 24 h and 1 year, respectively. This indicated that the anisotropic AgNPs gel still has antimicrobial activity even after storage for 1 year at room temperature.

**Figure 1.** Anisotropic AgNPs containing formulation. Gel without AgNPs (**a**), Gel containing 0.04 mg/g of anisotropic AgNPs stored at room temperature (27–30 ◦C) for 24 h (**b**) and 1 year (**c**).

**Figure 2.** Antimicrobial evaluation of anisotropic AgNPs gel. Antimicrobial test of anisotropic AgNPs gel toward MRSP MIC 411 after preparation for 24 h (**a**), and after storage at room temperature for 1 year (**b**). Inset: anisotropic AgNPs gel (i–iii), povidone iodine (iv), control gel (v).

### *3.2. Microbial Loads in Wounds*

To assess the antimicrobial activity of anisotropic AgNPs gel on wound infection, MRSP were infected with the initial inoculum of approximately 107–108 CFU/mL (Figure 3). The agents were applied after 48 h of infection. The results show that bacterial cells in

the group treated with anisotropic AgNPs gel and povidone iodine were significantly less than the control group on day 4 and day 7 (\* *p* < 0.05, \*\* *p* < 0.001). In the same way, these results show a continuous decrease of bacterial cells on day 0 to day 7 for groups that had anisotropic AgNPs gel applied and povidone iodine. On the other hand, the bacterial cells in the control group (Gel without AgNPs) continuously decreased from day 0 to day 10. These results exhibit that anisotropic AgNPs gel and povidone iodine can eradicate all the bacterial cells within 7 days, while the control group could eliminate all bacterial cells within 10 days.

**Figure 3.** Bacteria load average in wound area. MRSP MIC 411 was loaded into the wound at 107–108 CFU/mL. The bacteria were evaluated on days 0, 4, 7 and 10. Data represent the mean value ± SD (error bar) of 6 mice (*n* = 6). \* *p* < 0.05 and \*\* *p* < 0.001 are used as significant differences in comparison of the control gel with other groups.

### *3.3. Wound Closure*

We observed the wound healing activity of anisotropic AgNPs gel by quantitative measurement of the wound area on days 0, 4, 7, 10 and 14. The wound area on each day was compared with the initial wound area. Moreover, the wound healing was investigated by capturing digital photographs on days 1, 4, 7, 10, 14 and 21 of the experiment to evaluate wound features and scar formation. In Figure 4, wounds were made up to 8 mm in diameter, followed by adding bacterial cells at 107–108 CFU/mL. The results show that all groups had an efficiency of wound contraction, which appeared within 14 days. Within all groups, there was not a significant percentage of wound contraction on days 4, 7, 10 and 14. Similarly, digital photographs of wounds showed complete healing on day 14 for all groups. Interestingly, there was only one group that showed no visible scarring on day 21 (Figure 5) and that was the group treated with anisotropic AgNPs gel. From the foregoing, there was no difference in wound contraction observed among all groups within 14 days. However, scars were better in the group treated with anisotropic AgNPs gel. Moreover, it is noteworthy that on day 4 of wound creation, the control group revealed highly purulent wounds, while other groups did not show pus on wounds.

**Figure 4.** Wound contraction of experimental groups post treatment. Wound contraction (%) = ((wound area on day 0-wound area on day X)/(wound area on day 0)) × 100. Wounds were created 8 mm in diameter. After the wounds were created, they were left to infect with bacteria for 48 h. Control gel (without anisotropic AgNPs), povidone iodine, and anisotropic AgNPs gel were applied to the wounds on day 3 and continuously until the wounds had completely contracted. Wound size was measured on days 4, 7, 10, and 14 to calculate wound contraction. Data represent the mean value ± SD (error bar) for 6 mice (*n* = 6).

**Figure 5.** Photographs of wound repair at different times after excision of the wound model in mice. Effects of control gel (without anisotropic AgNPs), povidone iodine and anisotropic AgNPs gel on the healing of MRSP wound infection in mice. Representative images of mice from groups taken on days 1, 4, 7, 10, 14 and 21 after creation of the wound are shown. Mice were given a wound size 8 mm in diameter and bacterial cells added at 107–108 CFU/mL. After 48 h, the agents were applied to the wound.

### *3.4. Histology*

We evaluated histological sections of wound tissue on days 14 and 21 to support the healing properties of all groups. Hematoxylin and eosin were used to observe cells and tissue such as immune cells, fibroblast and collagen which implied wound status. Normal mouse skin was used as the control that indicated healthy skin. In Figure 6, normal skin showed an integrity of four layers, including epidermis (top layer skin), dermis (next to the top layer), sub-cutaneous fat (next to dermis) and muscle (deepest layer). In addition, there were some fibroblast cells inserted into the collagen fiber in the dermis layer. These are normal characteristics of mice skin. In other groups, including control gel (without AgNPs), povidone iodine and anisotropic AgNPs gel, mice tissue exhibited two layers of epidermis and dermis. They presented a lot of fibroblast cells on day 14. Moreover, the epidermis layer of all groups clearly appeared very swollen, when compared with normal skin.

**Figure 6.** Photomicrographs of histological section of wound tissue of mice. Histology of wounds on day 14, stained with hematoxylin-eosin. Normal tissue (**a**,**e**), control gel (**b**,**f**), povidone iodine (**c**,**g**), anisotropic AgNPs gel (**d**,**h**) at scale bar of 100× (upper row) and 50× (lower row).

On day 21, as shown in Figure 7, the fibroblast cells in all groups exhibited a decrease when compared to day 14. Moreover, collagen fiber can be observed more clearly. Interestingly, the group treated with anisotropic AgNPs gel displayed collagen fiber that was the most obvious and the most similar to normal skin. In contrast, groups treated with control gel and povidone iodine exhibited too-tight collagen fibers when compared to normal skin. This collagen formation is also related to the amount of fibroblast cells that showed in each group (Figure 7e,f). Such results are consistent with photographs of mice skin without scars on day 21 in the group treated with anisotropic AgNPs gel (Figure 5).

**Figure 7.** Photomicrographs of histological sections of mice wound tissue to evaluate collagen arrangement. Histology of wound tissue on day 21, stained with hematoxylin-eosin. Normal tissue (**a**,**e**), control gel (**b**,**f**), povidone iodine (**c**,**g**), anisotropic AgNPs gel (**d**,**h**) at scale bar of 100× (upper row) and 50× (lower row).

### **4. Discussion**

*S. pseudintermedius* is an opportunistic pathogen in most animal species and is an important pathogen in veterinary medicine. Furthermore, the bacteria is a zoonotic pathogen that can transmit to and infect humans, and infection of the bacteria in wounds results in delayed wound healing. In this study, we made a gel that contained anisotropic AgNPs to overcome wound infection. In our previous study, we have shown that anisotropic AgNPs gels have antibacterial activities and prolonged antibacterial activity against animal and human pathogens in vitro [35]. Moreover, anisotropic AgNPs have antimicrobial activity as good as silver nanospheres, but with lower cytotoxicity [35]. Therefore, anisotropic AgNPs were used for this study. In this study, we reported in vivo capabilities of anisotropic AgNPs that appear to promote bacterial killing and improve wound healing, when infected with MRSP in a mice model of a skin wound.

The gel containing anisotropic AgNPs had antimicrobial efficiency and could be stored at room temperature for a year without reduction of antimicrobial efficacy. This result is the same as in a previous study, where AgNPs gel has a good antimicrobial activity and low cytotoxicity [22,42]. To assess the antimicrobial and wound healing efficacy of anisotropic AgNPs gels in vivo, BALB/cAJcl mice were used as a model of wound infection. Mice were given a wound that was infected by methicillin-resistant *S. pseudintermedius*. After 2 days of wound creation, antimicrobial agents were applied and antibacterial, as well as wound healing, activities were determined. The results of antibacterial tests show that the groups treated with anisotropic AgNPs gel can eliminate bacteria within 7 days, the same as povidone iodine, while the control group (gel without anisotropic AgNPs) needed 10 days to remove all bacteria from the wound. Owing to there being no previous reports on the use of AgNPs or AgNPs gel against *S. pseudintermedius* infected wounds in vivo, we can only compare the antibacterial efficacy with the reported results of pathogenic bacteria such as *S. aureus* that share several features with *S. pseudintermedius* [4]. We found that anisotropic AgNPs gel can eliminate *S. pseudintermedius* faster than those observed on *S. aureus* in the past. For example, AgNPs can eliminate all *S. aureus* cells infecting the wound on day 21 after wound induction [40]. In another study, *S. aureus* infected a burn wound, and it was found that AgNPs eliminated bacteria within 17 days after the induction of the burn wound [43]; indeed, the different efficiency at eliminating bacteria might be caused by the bacteria's ability to cause disease. In this case, *S. pseudintermedius* MIC 411 mostly infects dog wounds and exhibits multidrug resistance [36]. These bacteria could contaminate a mice wound, but they might not cause infection similar to an infected dog

wound. As shown in Figure 3, the mice's immunity can eliminate bacteria within 10 days without applying antimicrobial agents. However, anisotropic AgNPs gel can support faster bacterial elimination. This point could be an advantage in the case of wound infection in which animals cannot eradicate bacteria by their immune system.

Wound healing efficiency was assessed by the measurement of wound contraction. The results show that all groups were not different on wound healing efficiency, with complete wound contraction within 14 days. In general, mice wounds of size 8 mm in diameter can heal themselves within 14 days [44]. As observed in this study, infection of wound with *S. pseudintermedius* might not delay the wound healing process as shown by the antibacterial test (Figure 3), that mice can remove all bacterial cells within 10 days. The reason is that *S. pseudintermedius* might contaminate the wound, but they cannot replicate and disrupt the wound healing process. Therefore, mice can remove bacteria by their immune system and the results show that wound contraction was not different among all groups; however, even though the wound healing efficiency of anisotropic AgNPs gel was not different from the control group, it was noteworthy that on day 4 of wound creation in the groups treated with anisotropic AgNPs gel, images showed reduction of purulent wounds, similar to the groups treated with povidone iodine, while the control group showed highly purulent wounds. This indicates that anisotropic AgNPs gel prevents pus on wounds, which might be caused by reduction of bacterial cells and inflammation similar to povidone iodine [45]. Our results were similar to a previous report by P. F. Myronov et al., (2019), that AgNPs play an important role in cleaning wounds from purulence by decreasing the microbial contamination and the number of neutrophils and white blood cells [46]. In addition, there are many previous research reports that confirm the wound healing activity of AgNPs by reduction of inflammation through cytokine modulation and inhibition of MMP-2 and MMP-9 [22,47,48]. In another study, AgNPs reduced the levels of the prototypic cytokines, tumor necrosis factor (TNF) and interleukin (IL-6) [49]. Moreover, AgNPs have shown decreased mitochondrial function and induction of apoptosis or apoptosis-like changes of cell morphology [50–52]. However, these points could be studied further in the future to confirm the wound healing action of anisotropic AgNPs.

Besides prevention of purulent wounds, it was clearly noticed in groups treated with anisotropic AgNPs gel that there was negligible scarring on day 21. That anisotropic AgNPs promote scarless wound healing, as found in our study, is similar to the study of Jun Tian et al. (2007); they have demonstrated that AgNPs act directly on dampening the process of inflammation, thus promoting scarless wound healing [47]. In addition, Jaya Jain et al. (2009) reported that, apart from antimicrobial activity, AgNPs inhibited MMPs and promoted scarless wound healing [22]. Moreover, V. Dhapte et al. (2014) proved that AgNPs presented scarless wound recovery without inflammation, because of effective cytokine modulation [53]. The scarless wound healing result corresponds to the histology result; mice given anisotropic AgNPs gel showed collagen fiber similar to normal skin on day 21. Collagen fiber is also important owing to the orientation and arrangement of collagen playing a key role during the remodeling phase, and consequently on the final scar appearance after wound closure. In this case, we could explain our result by the previous study of Karen H.L. Kwan et al. (2011), who pointed to the improvement of collagen properties of AgNPs that led to better fibril alignments in repaired skin, with a close resemblance to normal skin. AgNPs were predominantly responsible for regulating deposition of collagen, and their use resulted in excellent alignment in the wound healing process [54]. The collagen fiber alignment in this study supports scarless wound healing in the group treated with anisotropic AgNPs gel, and could be explained by scientific evidence since it had been previously studied. Nevertheless, the mechanism of collagen fiber alignment could not be elucidated in this study.

Based on the present results, we summarize the wound healing process of anisotropic AgNPs gel against wounds contaminated with MRSP, as illustrated in Figure 8. For the wound after 2 days of applying agents, the number of bacteria in the groups treated with anisotropic AgNPs gel and povidone iodine are reduced, and the wound exhibits

no purulence (Figures 3 and 5), whereas the control group shows purulent wounds and exhibits more bacterial cells than other groups. The prevention of purulent wounds in the groups treated with anisotropic AgNPs gel and povidone iodine could be caused by a decrease in bacterial cells. On day 7, all the bacterial cells were eliminated in the groups treated with anisotropic AgNPs gel and povidone iodine, while the control group still had bacterial cell contamination (Figure 3). Next, the wound shows complete contraction within 14 days for all groups (Figure 4). This indicates that anisotropic AgNPs gel might not promote the wound contraction and quicken the healing rate. Interestingly, in the groups treated with anisotropic AgNPs gel, the wound shows scar disappearance as a result of collagen alignment within 21 days (Figures 6 and 7), while the control group and povidone iodine group show scar formation. These imply that anisotropic AgNPs gel could affect the orientation and arrangement of collagen fiber.

**Figure 8.** Wound healing process schematic of anisotropic ANPs gel. The anisotropic AgNPs gel reduced wound purulence after applying gel for 2 days, whereas the control gel exhibited high pus formation. After that, anisotropic AgNPs gel can remove all bacterial cells within 7 days and there is complete wound contraction within 14 days. Finally, improving collagen alignment by anisotropic AgNPs gel shows scar disappearance within 21 days.

### **5. Conclusions**

Wound infection in animals such as dogs and cats with antimicrobial resistant bacteria, especially *S. pseudintermedius*, plays an important role in the delay of wound healing. In a previous study, we showed that anisotropic AgNPs have antimicrobial efficacy against animal and human pathogens, and also anisotropic AgNPs have low toxicity to human cells *in vitro*. Therefore, antimicrobial activity and wound healing activity of anisotropic AgNPs *in vivo* were evaluated in this study. Based on the results, even though anisotropic AgNPs gel might not promote the wound contraction and quicken the healing rate, we have proved that anisotropic AgNPs gel can eliminate bacterial cells on infected wounds and reduce pus, as well as showing collagen alignment in repaired skin with a close resemblance to normal skin. This is important evidence to support scarless wound healing in mice, and this research indicates that anisotropic AgNPs gel could be suitable for treating infected wounds. Our findings support the potent antibacterial activities and scar reduction of anisotropic AgNPs gel as antibacterial agents in infected wounds. These gels can prevent the proliferation and colonization of opportunistic bacteria, and finally promote natural wound healing along with reduction of scar occurrence.

**Author Contributions:** Conceptualization, S.T. and R.P.; methodology, S.T. and O.S.; validation, R.P., S.K. and N.P.; formal analysis, S.T.; investigation, S.T., O.S. and P.S.; resources, R.P. and S.D.; data curation, S.T.; writing—original draft preparation, S.T.; writing—review and editing, R.P. and N.P.; visualization, S.T.; supervision, R.P.; project administration, R.P.; funding acquisition, R.P. and S.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Research and Researchers for Industries (RRi) by the Thailand Research Fund (TRF), Bangkok, Thailand, grant number PHD59I0052.

**Institutional Review Board Statement:** The experimental protocol was approved by the animal ethics committee of Khon Kaen University, Khon Kaen, Thailand (Animal Ethics KKU 3/61).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Acknowledgments:** We would like to thank Research and Researchers for Industries (RRi) by the Thailand Research Fund (TRF), Bangkok, Thailand for supporting the scholarship. Many thanks to the Northeast Laboratory Animal Center, Khon Kaen University, Khon Kaen, Thailand for providing us with facilities for animal testing. The authors wish to thank our collaborator Prime Nanotechnology Co., Ltd. (Bangkok, Thailand) for kindly giving anisotropic AgNPs. *S. pseudintermedius* MIC 411 were kind gifts from Nuvee Prapasarakul, Department of Veterinary Microbiology Faculty of Veterinary Science, Chulalongkorn University. We would also like to thank Ian Thomas, a retired lecturer from the Department of Physics, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand, for the linguistic correction.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

### *Article* **Impact of Nano-Bromocriptine on Egg Production Performance and Prolactin Expression in Layers**

**Ahmed Dawod 1, Noha Osman 1, Hanim S. Heikal 1, Korany A. Ali 2, Omaima M. Kandil 3, Awad A. Shehata 4,5, Hafez M. Hafez 6,\* and Hamada Mahboub 1**

**Simple Summary:** Egg production is one of the most vital axes in the poultry industry. During the late laying period, the egg production continuously decreases, and pauses among the sequence of egg laying increases; however, the feed costs remain constant. Several attempts were carried out to improve the reproductive performance of laying hens by decreasing the prolactin level in the blood; an increase in this hormone initiates the onset of incubation behavior in chickens. In this study, we investigated the potential use of nano-bromocriptine to the improve egg production performance in laying hens. The use of alginate-bromocriptine leads to a significant reduction in the prolactin expression in the pituitary gland, which in turn allows the elongation in sequences and reduction in pauses, as well as the feed per dozen egg in laying hens. Further studies are needed to assess the impacts of nano-bromocriptine on other performance parameters. Thus, the improvement of egg production persistency must also go hand in hand with sustainable egg quality and the maintenance of the birds' health.

**Abstract:** The current study aimed to investigate the potential use of nano-bromocriptine in improving the laying performance of late laying hens by modulating the prolactin gene expression. A total of 150 NOVOgen brown laying hens aged 70 weeks were randomly allocated into three groups of 50 birds each. The first group was kept as a control, while the second and the third groups were treated with bromocriptine and nano-bromocriptine, respectively, at a dose of 100 μg/kg body weight per week. The pause days, egg production, feed per dozen egg, and Haugh unit were determined on a monthly basis. Also, the relative prolactin gene expression in the pituitary gland was quantified using qPCR and the number of the ovarian follicles was determined after slaughtering at the 84th week of age. It was found that nano-bromocriptine and bromocriptine improved egg laying performance with minimal pause days, reduced feed per dozen egg, and depressed the relative prolactin gene expression; however, nano-bromocriptine treatment was significantly effective compared to bromocriptine. In conclusion, nano-bromocriptine might be beneficial for elongating sequences and reducing pauses.

**Keywords:** prolactin; sodium alginate; nanotechnology; egg production; qPCR; chickens

**Citation:** Dawod, A.; Osman, N.; Heikal, H.S.; Ali, K.A.; Kandil, O.M.; Shehata, A.A.; Hafez, H.M.; Mahboub, H. Impact of Nano-Bromocriptine on Egg Production Performance and Prolactin Expression in Layers. *Animals* **2021**, *11*, 2842. https://doi.org/10.3390/ ani11102842

Academic Editors: Antonio Gonzalez-Bulnes and Nesreen Hashem

Received: 16 August 2021 Accepted: 24 September 2021 Published: 29 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

### **1. Introduction**

Although the global table eggs production has increased over the past decade to 76.7 million tonnes in 2018 [1], further improvement of egg production performance is urgently needed to fulfill the high demand for animal proteins. This highlights the urgent need to keep the laying persistency with sustainable egg production and quality that goes hand in hand with maintaining health and animal welfare, by considering the bird's physiology, nutritional requirements, management system, reproductive status, and breed selection. Although the extreme efforts which have been done to keep the persistency of egg production, a reduction in egg production accompanied by a deterioration of egg quality are usually common at or around 72 weeks of age [2]. During this period, the egg production continuously decreased and pauses among the sequence of egg laying increased with constant feed costs, causing huge economic losses.

Prolactin belongs to adenohypophysis hormones, is one of the most blamed factors accompanied by a progressive reduction of egg laying performance during the late laying period. This hormone is progressively increased by the time in plasma of late laying hens. It prevents gonadotrophin, which stimulates ovulation as well as estrogen production at the ovarian level [3]. The ovarian tissues in laying hens targeted the prolactin as it expressed the prolactin receptor mRNA [4]. So far, prolactin inhibits estradiol secretion in white ovarian follicles. However, it may improve or depress steroidogenesis in yellow ovarian follicles depended upon the level of prolactin dose, the type of the follicular layer secreting steroids, the order of the ovarian follicle in the ovulation hierarchy, and the phase of the ovulation cycle [5]. Increasing prolactin level during the active stage of laying is accompanied by the appearance of broodiness and cessation of egg production [6]. Several attempts were carried out to improve the reproductive performance of laying hens via decreasing prolactin using recombinant-derived chicken prolactin fusion protein or anti-vasoactive intestinal polypeptide serum to prevent broodiness in laying hens [7,8]. Others tried to depress prolactin chemically using a dopamine agonist, bromocriptine, an ergot derivative [9–11]. The release of prolactin from the adenohypophysis is governed by dopamine as it prevents the stimulatory effect of the vasoactive intestinal peptide on prolactin secretion. Therefore, bromocriptine as a dopamine agonist could be used to overcome the broodiness behaviors in laying hens [8]. Indeed, subcutaneous injection of bromocriptine in laying hens during the 17th to 36th week of life increases the egg production and depresses the prolactin production during the laying cycle up to the 72nd week of age [3].

Nevertheless, due to the low bioavailability of bromocriptine [12], there is a great demand for more convenient, effective, and safe ways for drug delivery. Nano-drug delivery systems could be a promising alternative to extend the half-life time of active principles and to sustain its delivery to the target sites [12–16]. It was found that nanocomposites reduced the dose of the drug and the desired biological activity could be obtained with minimal side effects. Therefore, the present study aimed to synthesize, and characterize, alginate-bromocriptine nanocomposite as well as to assess its efficacy on egg laying performance during the late laying stage. Moreover, its modulatory effect on the prolactin hormone gene expression in the pituitary gland was studied.

### **2. Materials and Methods**

*2.1. Preparation of Alginate-Bromocriptine Nano-Composite*

The alginate-bromocriptine nano-composite was prepared according to Siddique et al. [12]. Briefly, 100 mg of 2-Bromo-α-ergocryptine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 5 mL ethanol and added portion wise to sodium alginate solution, previously prepared by dissolving 0.1 g of sodium alginate (Fisher Scientific, Waltham, MA, USA) in 100 mL distilled water with stirring. The addition process was performed drop by drop over 30 min with stirring and worming at 40 ◦C. The mixture was then heated at 50 ◦C under ultra-sonication at 100 W with 35 kHz for 45 min. The obtained nano-composite solution was air-dried then stored at 4 ◦C, dry, and dark conditions.

The formation and the morphology of alginate-bromocriptine nano-composite were characterized by transmutation electron microscopy (TEM, JEM-2100, JEOL- Tokyo Japan) and the average size of the prepared nano-composite was estimated. The Fourier Transform Infrared Spectrophotometer (FTIR) spectra were analyzed using a spectrometer (JASCO, Tokyo, Japan) in the range of 400–4000 cm−1. Potassium bromide discs (KBr) (5 mg of particles, 100 mg KBr pellets) were used as reference material. The optical properties of the bromocriptine and alginate-bromocriptine nanocomposite were investigated by measuring the UV-visible spectrophotometer (JASCO spectrophotometer, Tokyo, Japan) in the range of 200–800 nm.

### *2.2. Experimental Assessment of the Efficacy of Bromocriptine and Alginate-Nano-Bromocriptine* 2.2.1. Ethical Approval

All procedures of the experiment were conducted under the ethical guidelines of the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, University of Sadat City, Egypt (Ethical approval number: VUSC-017-1-19).

### 2.2.2. Experimental Design

One hundred fifty laying hens of NOVOgen brown strain of 70 weeks age were selected from a local commercial layer farm in Egypt based on good feathers, body weight (mean = 1800 ± 150 g), and free from any abnormalities. Birds were randomly allocated into 3 groups of 50 birds per each. Hens were allowed to have 2 weeks acclimatization period. Chickens in the first group were treated with saline and kept as control, while chickens kept in the second and third groups were treated with bromocriptine (2-bromo-alphaergocriptine, Sigma-Aldrich, St. Louis, MO, USA) and the prepared nano-bromocriptine, respectively, at a dose of 100 μg/kg BW/week according to Reddy et al. [17] for 12 weeks (from 72nd to 84th week of age). Each group was subdivided into two subgroups; the first subgroup was treated orally, whereas the second subgroup was treated subcutaneously underneath the wing. Hens were kept under the standard laying conditions on the litter floor system of sawdust equipped with automatic drinker and feeding systems. The feed containing 16% crude protein (corn-soybean meal of 16% CP, 2900 kcal ME/kg diet) and 120 g/hen/day was used, fresh-water give at ad libitum. The photoperiod was kept at 16 h of light/day, achieved via an automatic lighting system. The temperature of the house was kept at 24 ± 3 ◦C.

### 2.2.3. Egg Production Performance

Eggs were collected and recorded daily from each group at 12 AM. Pause days, egg production%, feed intake, and feed per dozen eggs were daily evaluated for each subgroup. Pause days were daily estimated for each laying hen as the hens were separately reared in deep litter system. Also, twelve eggs were randomly selected from each group/subgroup at 76th, 80th, 84th/week of age to determine the Haugh unit according to Haugh [18] using the formula: Haugh unit (*HU*) = 100 × *log* - *H* − 1.7*W*0.37 + 7.6 , where "*H*" is the albumen height and "*W*" is the egg weight.

At the 84th week of age, all hens were slaughtered by manual cervical dislocation then dressed. The ovary was investigated to estimate the number of normal large yellow follicles (LYF) (>10 mm diameter), small yellow follicles (SYF) (5–10 mm diameter), and large white follicles (LWF) (3–5 mm diameter) according to Renema et al. [19].

### 2.2.4. Prolactin Gene Expression in Pituitary Gland Using qRT-PCR

Just after slaughtering, the pituitary gland was collected randomly from 5 hens/subgroup. The RNA extraction was carried out using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer and the cDNA was formed from total RNA using Maxima First Strand cDNA Synthesis Kit (Life Technologies, Carlsbad, CA, USA). The PRLE2F: 5 -GTTTGTTTCTGGCGGTTC-3 , and PRLE2R: 5 -AAATTCATTGAATATTTCTGAAG-3 primers were used to amplify 181 bp fragment of prolactin gene [20], while the QGAPDHF: