Cellular and Molecular Biology ZnO, TiO 2 and Ag nanoparticles impact against some species of pathogenic bacteria and yeast

: The economic approaches for manufacturing the nanoparticles with physical and chemical effects and limited resistance to antibiotics have been pro gressed recently due to the rise of microbial resistance to antibiotics. This research aimed to study the antimicrobial efficacy of silver nanoparticles Ag, ZnO, and Tio2 nanoparticles against Salmonella typhimurium and Brucella abortus and Candida albicans . Two isolates of Salmonella and two isolates of Brucella abortus were isolated from food spastically meat and blood specimens, respectively. Candida albicans were isolated from the patient's mouth with oral candidiasis (oral thrush) and confirmed diagnosis by API 20C test. The antimicrobial susceptibility of Salmonella typhimurium and B. abortus isolates were performed against nine different antibiotics. Silver nanoparticles consisting of AgNPs size (90) nm, ZnO NPs size (20, 50) nm as well as TiO2 NPs size (10, 50) nm, were used. UV-Visible spectrophotometer was used to characterize silver nanoparticles. The highest resistance of Candida albicans was seen for fluconazole, Clotrimazole and Itracona -zole. The results of the Minimum Inhibitory Concentration (MIC) of nanoparticles against Salmonella typhimurium showed the average MIC of Tio 2 -10nm and Tio 2 -50nm were 5000 and 2500 μg\ml for S1 and S2 isolates, respectively. The isolated Brucella abortus (B1 and B2) showed sensitivity to NPs with different MIC. The average MIC for Ag-90nm was 5000 and 2500 µg/ml for B1 and B2 isolates, respectively. The findings suggest NP solution has fungicidal and bacteri cidal impacts on the tested microorganisms so they can be suitable for multiple of the biomedical field such as developing new antimicrobial agents.


Introduction
The increasing bacterial resistance to multiple antibiotics has raised the demand for alternative antimicrobial agents. In this regard, to avoid antibiotic use, scientists have been interested in nanoparticles, a nanoscale material ranging from 1-100 nanometers (1). The metal oxide nanomaterials possess antimicrobial activity over a wide range of both Gram-positive and Gram-negative bacteria, including resistant bacterial strains. Iron oxide (Fe 3 O 4 ), zinc oxide (ZnO), magnesium oxide (MgO), copper oxide (CuO), and titanium dioxide (TiO 2 ) nanoparticles are well-known nanomaterials with high antimicrobial properties (2). This potency of antibacterial activity might be attributed to the generation of reactive oxygen species (ROS) as their intrinsic photocatalytic activity or the metallic ions release (3).
According to World Health Organization (WHO) report, non-typhoidal Salmonella represents one of the major etiological agents of infectious diseases worldwide (4). Multidrug resistance non-typhoidal Salmonella emergence worldwide is a critical public health issue, for which severe problems would be generated for the treating intestinal and extra-intestinal infection complications (5). Multidrug resistance is associated with enhanced virulence due to the rapid global spread of these multidrug-resistant non-typhoidal Salmonella strains among human societies (6). Additionally, based on research conducted in Denmark, the mortality rate was reported higher in patients who have an infection due to multidrug-resistant Salmonella typhimurium compared to those caused by sensitive strains (7). Surprisingly, there is a discriminating advantage for resistance Salmonella in the environment where extra amounts of antibiotics are used because the antibiotic treatment itself acts as a risk factor for infection with the resistant bacteria (8).
Antibiotics with penetrating ability to macrophages are preferred for brucellosis treatment. Consequently, brucellosis relapsing is seen not due to anti-Brucella drug resistance but because of microorganisms' ability to last inside host cells away from the antibiotics. Thus, antibiotics with intracellular activity are considered effective tools in managing brucellosis (9). As a result of increasing therapeutic failure, relapses, toxicity, and side effects of the conventional antibiotic regimen, searching for new medications has been raised during recent years (10).
Candida is a ubiquitous eukaryotic yeast residing in healthy individuals as a part of the normal microbial flora of the skin, gastrointestinal tract, and genitourinary tract. The risk of developing opportunistic Candida infections increases when host immunity is compromised. The most common species associated with Candida in-fections is Candida albicans (11), often manifested as oral thrush, vulvovaginitis cutaneous candidiasis (12). National Committee has published recent guidelines for macro-and microdilution assay for in vitro testing of antifungal susceptibilities for Clinical Laboratory Standards (NCCLS) (13). These tests showed a well in vitro correlation (14).
Nano antibiotics (nanomaterial possessing antimicrobial properties) could act as an alternative to fighting against multi-drug resistant organisms (15). Silver nanoparticles (Ag NPs), because of their antiviral and antibacterial properties, are known as the most promising nano-antibiotics (16). For several decades' silver has been used as a disinfectant agent. Inclusively, silver nitrate has been used for treating burns and infection control. However, due to the discovery of penicillin, the use of silver as an antimicrobial agent has been reduced. Currently, the increasing resistance to antifungal agents has attracted attention to silver as an antibiotic once again.
Among nanosized metal oxides, zinc oxide (ZnO), due to its attractive properties such as high surface-tovolume ratio, low cost, and long-term environmental stability, has gained much attention (17). Several studies have already reported that ZnO nanoparticles are non-toxic to human cells and toxic to bacterial cells. Toxicity studies showed that DNA in human cells is not damaged by zinc ions; a property that makes ZnO nanoparticles biocompatible with human cells.
The aims of this study were the isolation of (Salmonella typhimurium, Brucella abortus, and Candida albicans) from food and different clinical specimens, and identification of the isolated bacteria by rapid method serological, via VITEK2 systems. In addition to assessing the activity of conventional antimicrobial activity against these different pathogens compared with the effects of nanoparticles in different sizes (ZnO and TiO 2 and Ag) with an antibiotic-resistant pathogen.

Isolation and bacterial identification (Salmonella typhimurium and Brucella abortus)
Two isolates of Salmonella were isolated from food spastically meat. The enrichment phase in the selective liquid environment was carried out using Rappaport Vassiliadis Broth, which is considered as a selective enrichment for detecting Salmonella spp. from foods (18). The goal of this step was to increase the growth and vitality of the bacteria. Rappaport Vassiliadis (V.R.) broth was used through the transfer of 0.1 ml of the second diluents to a tube containing 9 ml of sterile broth and incubated for 18-24 hrs in the presence of 5-10% CO2. At 37 °C , 0.1 ml of the broth was transferred to the surface of XLD media and incubated for 18-24 h. The two isolates of Brucella abortus were isolated from blood specimens and cultured using a BACTEC automated blood culture system (9050; Becton & Dickinson, Franklin Lakes, NJ, USA). The specimens were incubated at 37 °C for 7 to 30 days and were sub-cultured in Mueller Hinton plus 5% blood and Brucella agar plates. Rose Bengal and 2-mercaptoethanol (2-ME) agglutination serological tests were performed to detect Brucella antibodies. In addition to the Conventional Biochemical tests, the VITEK-2 system (19) [bioMe 'rieux] was used according to McFadden (20).
2-Isolation of fungi: fungi, especially Candida albicans, were isolated from patients' mouths, who had oral candidiasis (oral thrush) by sterile swabs containing transported media. After inoculation on Sabouraud's Dextrose agar and incubation for 48hrs at 37 o C, in addition to the API 20C test (21) (Merieux Bio) and germ tube were used to them. Oral Candida colonization is usually displayed through a microscopic examination using potassium hydroxide (KOH) or fungal cultures.

Susceptibility to antibacterial agents
The antimicrobial susceptibility of S. typhimurium and B. abortus isolates were performed against nine different antibiotics (22). For this purpose, the disc diffusion method was recruited according to the Clinical and Laboratory Standards Institute instruction (23). The antibiotic discs used in this study were (Becton Dickinson Co, Maryland, USA): Ampicillin (AM/10 µg), Gentamicin (G.M./10 µg), Chloramphenicol (C/30 µg), Amikacin (AK/30 µg), Nitrofurantoin (NIT/300 µg), Rifampin (FEP/30 µg), Imipenem (IPM/10 µg), Ceftriaxone (CRO/30 µg) to evaluate antimicrobial susceptibility for both two strains of each S. typhimurium and B. abortus respectively Table1. The discs impregnated micropipette and withdrawing grown to several times (6)(7)(8) and this made any weakness diluted concentration equivalent to 2500 micrograms/ml. 3. 100 ul of well 1 was transformed to well two and blended the same way to form a dilution with a concentration of 1250 ug/ml. 4. Repeated process down to well No. 10 and then pull them 100 ul and neglected and thus we get the concentrations (625,312.5, 156. 25, 78.125, 39.06, 19.53, 9.76 ug/ml). 5. The micro-titer plate was covered and incubated at 37 0 C for  hours. 6. The MIC of nanoparticles is determined by the lowest concentration that withdrawn the observable growth of bacteria. The indication of microorganism's growth in control tubes was examined by tube turbidity.

Agar well-diffusion method
The standard agar well diffusion method was used to investigate nanoparticles' antibacterial effects (28). For culturing the bacteria and fungus, Mueller-Hinton agar medium and potato dextrose agar medium were used respectively to test antimicrobial activity by agar well diffusion method against pathogenic microorganisms S. typhimurium, B. abortus, and C. albicans. The pure cultures of pathogens were prepared in 10 ml of nutrient broth followed by overnight shaking at 37 o C. Each strain was swapped homogeneously onto the individual Muller-Hinton and potato dextrose agar using sterile cotton swabs [CLSI, 2009:29(3)]. The six wells by sterile, boring cork (5 mm diameter) puncture. The 5 mm well was impregnated with different concentrations of each Ag 90, ZnO 20,50, NPs, and TiO 20,50. For making No.6 to well negative control, distal water was added to it and incubated at 37°C for 24hrs. The process was repeated three times. To determine the inhibitory growth of the NPs on the specific pathogen, the mean value was taken into consideration (29).

Antifungal disk paper preparation
All antifungal drugs were obtained in powdered form. A stock solution of each drug using dimethyl sulfoxide (except fluconazole in sterile double distilled water) was prepared by applying 20µl of an appropriate dilution of the test solution to 6 mm in diameter of Whatman filter paper No.1 (30) as follows: Fluconazole (10 µg), Ketoconazole (30 µg), Clotrimazole (10 µg), Voriconazole (1µg), and Amphotericin-B (20 µg). According to the antifungal potency of (Rosco Diagnostica company), they were taken out and allowed to dry under aseptic conditions and stored at °C in a refrigerator.

Antifungal susceptibility testing
The disk dissemination method was used to test antifungal susceptibility. As the "CLSI" (M44-A) procedures, the inoculum was set up by picking five particular colonies from a 24 hr old Candida species culture. Colonies were suspended in 5ml of sterile 0.9% normal saline. The suspension was vortexed to get uniform turbidity and adjusted visually to 0.5 McFarland standards. The suspension was injected into "Mueller Hinton Glucose Agar containing 2% glucose. Moreover, 0.5 µg/ml of methylene blue was used for susceptibility testing. Antifungal disks are placed so that they are not closer than 24 mm from the center. The plates were located in an incubator at 37 °C for 24 hr. The inhibition zone with antibiotics were placed using sterile forceps. The effect of the antibiotics was tested in each dish. Then the petri dishes were incubated bottom up for 18-24 h at 37 °C. The results were assessed by the presence of inhibition zones around the disks. Test organism growth inhibition at a distance from the disc with the antibiotic was indicative of the strain's susceptibility. If the test organism developed close to the disk with antibiotics, this organism was determined as resistant to the antibiotic CLSI 2006, M 100-S16).

Preparation of nanoparticles
Silver nanoparticles consisted of AgNPs size (90 nm), ZnO NPs size (20,50) nm as well as TiO2NPs size (10,50) nm, were used. The M. K Impex Corporation-Canada Company has set some qualities to manufacture silver nanoparticles. The particles should be measured at 20 nm with a molecular weight of 107.87 g / mol. The density should be 10.49 g / ml with a purity of 99.95%. Powder grey color, and shape of spherical product chemical way and space superficial density of 20 g/m 2 but silver nanoparticles measuring 90 nm with a purity of 99.9 % and the item is received from the company in the form of plastic explosive 25 g capacity. ZnO N.P.s was in the form of powder, reaching the size (20) nm and purity 99.9% white color powder, molecular weight 81.39 g / mol density 5.610 g / ml. The company has prepared multiple plastic containers containing one of which 100 g, and for the TiO 2 NPs measuring 10 nm has been received in the form of nanopowder, measuring nanoparticles as 10nm and an area of the shallow grave of 225 g / m 2 , the degree of purity of 99%, moisture 0.48%, TiO 2 NPs measuring 50 nm and a degree purity of 99%.
According to the method, a stock solution was prepared (24). One hundred mg of nanoparticle powders added to 10 ml of deionized water next shaking vigorously to break the mass for a homogeneous solution in 5 minutes and then sterilized at 121 0 C for 20 minutes, cooled at room temperature to get a final concentration of stocks 10 mg/ml.

UV-visible spectrophotometer
UV-Visible spectrophotometer (Thermo Scientific-NanoDrop1000 lab tech international) was used to characterize silver nanoparticles. The Scientific research center/Salahaddin University's device at 400-800nm helped characterize silver nanoparticles (25).

Antimicrobial activity of nanoparticles
Broth Dilution Method: A prerequisite way used for quantitative assessment to demonstrate the impact the effectiveness of nanoparticle using microtiter plate 96 holes, and conducted depending on the method as follows (27): 100 ul of Mueller Hinton broth was added to each well. 2. 100 ul of nanoparticle prepared at concentration 5000 ug /ml was added to No. 1 well within the pit row A. has been blending the counter through the use of diameter was measured in millimeters using a ruler for a separately antifungal disk. Clarification of entirely antifungal susceptibility (Susceptible S and resistant R) was made according to (CLSI M44A standards, 2009) ( Table 2).

Results and Discussion
The automated VITEK 2 compact system was the final step to identify Brucella abortus using Gram-negative bacteria code according to the manufacturer's instructions, which checked 47 biochemical tests and one as a negative control thoroughly. B. abortus differentiated by their requirement to CO2, H 2 S production and positive in ILATk (L-lactate alkalization), the food samples were examined using Rappaport Vassiliadis Broth found non-typhoid Salmonella, it enriches salmonellae especially S. typhimurium because they can multiply at relatively lower pH compared to other gut bacteria. Rappaport Vassiliadis Broth has a p.H. around 5.2. Also identified by their ability to grow, colonies may range from clear to pink /red. Most colonies 2-4 mm with black centers on XLD agar ferment glucose and produce (gas, H 2 S). For obtaining the sera, both of the clotted blood samples were centrifuged at 3,000 × g for 10 min, and 2-mercaptoethanol (2-ME) agglutination tests were performed to perceive Brucella antibodies. Titers higher than 1:40 were considered positive for 2-ME tests based on reference. The primary commercial product used for testing medically significant yeasts was the original API 20C yeast identification strip with index code. One of the reactions which gave results for all the isolates tested were: glucose (+), 2-keto-glutarate (+), inositol (-), cellobiose (-), lactose (-), maltose (+), raffinose (-), and galactose (+). Along with other morphological features, a creamy round colony on SDA allowed the identification of Candida species. The reactions were confirmed by the germ tube after incubating Candida with 0.5ml human serum for 3 hrs at 37 °C, and 10% KOH showed positive.
This study generally demonstrated the antimicrobial susceptibility of S. typhimurium and B. abortus isolates against nine different antibiotics. The results indicated B. abortus as the most multidrug resistance isolated bacteria that phenotypically showed sensitivity to only three antibiotics, including Ampicillin, Chloramphenicol, gentamicin, and represented resistance to six antibiotics Cefoxitin, ciprofloxacin, imipenem, and rifampicin Amikacin respectively. At the same time, isolates of Salmonella were resistant to carbapenems (Imipenem), β-lactams (cefotaxime, Ceftriaxone), aminoglycosides (Amikacin), and Gentamycin. Salmonella typhimurium and Brucella abortus were resistant to β-lactams (Ceftriaxone, Cefoxitin), aminoglycosides (amikacin), imi-penem and susceptible to Gentamycin and Chloramphenicol, as presented in Table 1. These results align with the growing rates of antimicrobial resistance among non-typhoidal Salmonella, highlighting the caution in using these antibiotics (31).
Standard first-line therapy for Salmonella infections is based on Chloramphenicol, sulfa drugs, Ampicillin, and fluoroquinolone. In this case, all of these antibiotics would be unsuccessful, if the Salmonella was a multidrug-resistant strain, and treatment will be brutal. A former statement designates death in the patients subsequently, a nosocomial Salmonella epidemic in a U.S. hospital (32).
Fluoroquinolones, trimethoprim/ sulfamethoxazole, and Ampicillin are well-known as typical first-line therapy for Salmonellosis. The emergence of resistance to these antibiotics in non-typhoidal Salmonella represents a severe problem in treating infections due to these organisms (33). A discriminating advantage could be wellthought-out for resistant Salmonella in the environment where unnecessary antibiotics are used, and the antibiotic therapy itself is an important risk factor for infection with resistant bacteria (8).
A rapid increase has been seen in microbial resistance to common antibiotics, signalizing the urgent need for new antibiotics classes to treat many infectious diseases. The WHO declared that only a few antibiotics are clinically efficient and have good intracellular penetration for brucellosis treatment (34). The results revealed that both B. abortus isolates were phenotypically resistant to Cefoxitin, Amikacin, Nitrofurantoin, Imperium, and Rifampicin. These results are inconsistent with those reported by Gattringer et al. (2010), who found out that REP Rifamycin had remarkable bactericidal activity at a low concentration (35). Bacterial RNA and protein synthesis could be blocked by Rifamycins and result in a noticeable bactericidal effect inside and outside the cell. However, some patients are prevented from using RIF for an extended period because of the apparent gastrointestinal reaction. Yet, an alternative medication or combined use of additional drugs should be used to reduce patients' discomfort.
Indeed, the excitation of several metabolic processes can also contribute to rifampin resistance in Brucella (36). This study showed variable antibiotic susceptibility of the bacterial isolates to gentamicin, Ampicillin, Ceftriaxone, and, Chloramphenicol. As the most effective antibacterial agent (37), Ceftriaxone is described with a high outcome on bacteria in idiophase and mostly performances on the bacterial cell wall. Still, it does not affect the ongoing cell wall synthesizing in the inactive condition. While there are only preliminary case reports on the handling of Brucella infection by gentamicin or Ampicillin (38), AMP, and aminoglycosides medica-
The scientific community, like the practitioners of the food industry, has provoked the hunt for antimicrobial alternatives to fight the ongoing development of bacterial strains resistant to multiple antibiotics. Silver, often regarded as a broad-spectrum antimicrobial agent, may therefore be a suitable option, particularly in its nanoform (41). The various N.P.s were talented to constrain the bacterial progress at diverse concentrations. The MIC in both methods (micro-dilution and well diffusion methods) for Ag 90nm were 2500 μg\ml 13.5mm and 2500 μg\ml 12mm. It indicates that silver nanoparticles possess antibacterial action against Salmonella typhimurium as shown in Table 3. Kumar et al (2012) reported an excellent antimicrobial activity of silver nanoparticles (10-100) nm against five clinical pathogenic bacteria (S. typhii, E. coli, Klebsiella pneumoniae, S. aureus, and Vibrio cholerae) (42). Silver nanoparticles are well known for their antimicrobial activities against a wide range of pathogenic organisms (43).
Contrary, moderate antimicrobial action was observed in the S. typhi (44), which agrees with the results of the present research. On the other hand, AgNPs are an effective antibiotic against S. epidermidis, S. aureus, E. coli, and S. typhi (45). In contrast, Zarei et al. (2014) reported that Ag NPs displayed antimicrobial activity against a single strain of S. Typhimurium grown in tryptone soya broth at 35C, reporting a MIC of 3.12mg/mL and an MBC of 6.25mg/mL (46); the values of MIC and MBC are lower than those reported in the present study. This may, in part, be due to the lesser size of the Ag NPs used in their study (10 nm) compared to the average size in this study (20 nm) or differences in NP Dissolution behavior.
The silver nanoparticles showed a maximum absorption band at 430 nm, indicating that the nanoparticles were scattered in the aqueous solution, preventing the AgNPs from agglomeration in UV-Vis ( Figure 1). UV-Vis profile of AgNPs exposed a peak at 400 nm, which matches the typical surface plasmon resonance of AgNPs. The ZnO nanoparticles' absorption band demonstrated a blue shift (from 275 nm to 350 nm) due to the quantum confinement of the excitons present in the sample. The quantum size effect of these nanoparticles is indicated by this optical phenomenon shown in Figure 2. Figure 3 represents the FT-IR spectrum of TiO 2 . Various characteristics peaks are shown. The absorption band range was 3454.51cm-1, and it is related to  Figure 1 demonstrated the UV-Visible spectra of Silver nanoparticles. U.V. visible spectra were recorded from the reaction medium, which U.V.-vis spectrophotometer characterized. The silver surface Plasmon resonance band has been seen at 426 nm, and the silver surface Plasmon resonance band appears at 426 nm.

Nanoparticle
Bacteria Isolates S1 S2   stretching. 1631.78 cm-1 to bending O-H vibration, demonstrating the water as moisture. The intense characteristics peak of TiO 2 was seen at 690.52 cm-1, and it is also assigned to the Ti-O stretching band (47). Fig.4. The spectrum shown in the figure demonstrated a broad peak around 457 cm¡1 and shoulder around 545 cm¡1, corresponding ZnO nanoparticlescomparatively, and other ranges showed a smooth peak. Figure 5 shows Fourier transform-infrared spectra of silver nanoparticles. FTIR spectral absorption peaks were at 773, 800, 813, 1040, 1288, 1530, and 3226/ cm in the region 500-4000/cm ( Figure 5). The biomolecules and their different functional groups have been seen through FTIR analysis. Figure 6 well-defined the optical absorption spectra of TiO 2 nanoparticles. TiO 2 nanoparticles appear transparent in the visible region. A sharp absorbance peak around 2.93 eV has also been observed. The value n=1/2 does not appear to be meaningful for data of the bandgap energy, indicating that TiO2 is a direct bandgap type semiconductor.
Numerous mechanisms have been suggested to elucidate the antibacterial properties of ZnO nanoparticles. The formation of hydrogen peroxide from the surface of ZnO is considered an effective means of inhibiting bacterial development (50). The release of Zn2+ ions, which prefer to disrupt the cell membrane lipids and proteins, resulted in the leakage of intracellular contents and, eventually, cell death is another possible mechanism for ZnO antibacterial activity (51). Other researchers approve the important antibacterial activity of ZnO nanoparticles wherein the nanoparticles might entirely lyse S. typhimurium and S. aureus, causing food poisoning (52). In a study carried out by Duffy et al.
(2018), the MIC was reported for the five Salmonella strains (312.5 μg\ml 625 μg\ml), indicating lower effective treatment of Salmonella than our study (53). Tayel et al. (2011) used ZnO NPs of similar particle size (50 nm)to the current study, the nutrient broth was the incubation media used for the S. Enteritidis, and S. Typhimurium strains (54). However, their results were lower than that ours.
TiO 2 nanoparticles are interesting subjects for microbial studies because of their specific characterization as size, shape, and structure of Tio 2 nanomaterials crystal, surface stability, and the transferences between different    phases of TiO 2 under stress and heat. Tio 2 Nano-materials were known as the glamour of recent medical research due to their microbicidal effect on other diseasecausing organisms (56). The bactericidal effect of TiO 2 nanoparticles on bacteria is of extreme importance due to pathogenic bacteria's ability to enter an ecosystem's food chain (55,57). Mutagenicity evaluation of metal oxide nanoparticles by the bacterial reverse mutation assay.
If the concentration of Tio 2 increases, the inhibition zone also increased as per the measurements described by the inhibition zone. Nanomaterials are well known to show strong inhibiting effects against a wide range of bacterial strains. According to numerous studies, it is believed that the metal oxides convey the positive charge while the microorganisms have negative charges; this causes electromagnetic attraction between microorganisms and the metal oxides, which leads to oxidization and, finally, microorganisms' death (57).
The isolated Brucella abortus (B1 B2) showed sensitivity to NPs with the different MIC (5000 µg/ml) for Ag-90nm and inhibition zone of 12 mm. However, with a MIC of 2500(µg/ml), the inhibition zone was 13.5 mm. The ZnO-20nm MIC of 156.25(µg/ml) had an inhibition zone of 15.5 mm, and the MIC of 312.5(µg/ml) had an inhibition zone of 18 mm. Also, the ZnO-50nm MIC of 625(µg/ml) had an inhibition zone of 10 mm, and the MIC of 1250(µg/ml) had an inhibition zone of 11 mm. TiO 2 -10nm with MIC of 5000(µg/ml) had an inhibition zone of 12 mm, and MIC of 2500(µg/ml) had an inhibition zone of 11 mm. Nevertheless, TiO 2 -50nm with MIC of 2500(µg/ml) had an inhibition zone of 11 mm, and MIC of 2500(µg/ml) had an inhibition zone of 13 mm, as shown in Table 4.
Numerous studies demonstrated that AgNP activity is proportional to its size (57,58). It was found that AgNPs of smaller dimensions can penetrate bacteria (59). Our result revealed that silver nanoparticles have an instant antimicrobial impact on intramacrophage B. abortus. A study recorded that silver nanoparticles have an antimicrobial impact on intramacrophage B. abor-tus 544 (60). MBC, MIC, and experiments at two ppm and one ppm of silver nanoparticle concentrations have shown that silver nanoparticles have an antimicrobial effect against intramacrophage B. abortus 544 over 24 hours and have shown that silver nanoparticles have an antimicrobial effect against intramacrophage B. abortus 544 and may therefore be useful for brucellosis care.
In the current study, ZnO nanoparticles prevented the growth of bacterial isolates. The tested isolates are vulnerable to more than the MIC of ZnO nanoparticles than high resistance to multiple routine antibiotics classes. Jiang et al. (2008) described the antimicrobial action of ZnO nanoparticles against food-related bacteria (61). Various mechanisms have been described for the antimicrobial activity of ZnO nanoparticles. The hydrogen peroxide generation and Zn2+ ions release have the ability of cell membrane and intracellular contents damage. Also, by reducing the particle size of nanoparticles and increasing surface area, the antimicrobial properties of the ZnO will rise. Haghi et al. (2012) has shown that 150 mg/ml of TiO 2 and 200 mg/ml of TiO 2 have the best effect on this study's seven bacterial isolates, while 50 mg/ml of TiO 2 and 100 mg/ml of TiO 2 have a little antibacterial effect on half of them (62).
TiO 2 nanoparticles with 1-100nm diameter are interesting to investigators because of specific properties such as size, shape, and structure of TiO 2 nanomaterials crystal, surface stability, and the transferences between different phases TiO 2 under stress and heat. TiO 2 Nanomaterials were known as recent medical research glamour due to their microbicidal effect on other diseasecausing organisms (56). The bactericidal effect of TiO 2 nanoparticles on bacteria is of great importance due to pathogenic bacteria's ability to enter an ecosystem's food chain (57). The minimum inhibition concentration (ug/ ml) and inhibition zone (mm) of antibacterial activity of nanoparticles against Brucella spp have been shown in (Table 4). In the expansion of novel nanodevices which can be used in many physical, biological, biomedical, and pharmaceutical applications nanosized inorganic   particles are recognized as a progressively important material (63). The MIC and MFC values varied between 2500 µg/ml and 625 µg g/mL and showed inhibition zone ranged 11-15,5 mm respectively as in Table 5. This research investigated the antifungal activity of nano-Ag against C. albicans. Antifungal activity of Nano-Ag towards fungal strains compared to amphotericin B's antifungal activity in the zone inhibition values is also tested. Kim et al. (2009) reported that nano-Ag has remarkable antifungal potency in treating fungal infectious diseases (64). Herrera et al. (2001) represent nano-Ag as a proper antimicrobial agent, mainly because it is highly toxic to most microorganisms (65). Endo et al. (1997) have reported that the inhibition of bud growth relates to membrane damage (66). This report suggests that probably due to membrane integrity destruction, nano-Ag could inhibit the normal budding process.
the results obtained in this study demonstrate the importance of the N.P.s size, as the lowest nanoparticles bring the highest inhibition (67). Lara et al. (2015) presented that biofilm uncovered to AgNPs shows a few Candida albicans filamentous formations (68).
There is incidental evidence that these particles have good antimicrobial activity (69). The AgNPs have shown multiple antimicrobial activities against a wide array of microorganisms, possibly because of multiple mechanisms involved in its antimicrobial action, including activity against drug-resistant bacteria, fungi, and viruses (70). Since significant differences in terms of sufficient size and concentration, the action was observed for the three investigated pathogens. This may be attributed to many features strictly related to each strain's genetic features, including the presence of specific genetic determinants of resistance (71). Fayaz et al. (2012) showed that the AgNPs-coated condom has antiviral, antibacterial, and anti-fungi (against Candida spp.) properties (72). This suggests that AgNPs can treat all multi-drug resistant pathogens of diverse types from all clinical sources.
According to the findings, the AgNPs solution has fungistatic and fungicidal effects against the microorganism tested. It also suggests that it can be suitable to develop new antimicrobial agents with multiple biomedical field applications. The inhibition zone formed around C. Albicans was slightly further than that of the other two organisms when AgNps was used alone.
The fungicidal effects of ZnO-NP treatments, such as ZnO nanocomposites and ZnO, have recently been established (28). ZnO-NP has also been reported to exhibit inhibitory effects on bacterial biofilm formation and candidal biofilm formation (73). Consequently, evaluated the antimicrobial effects of ZnO on C. albicans) MICs of ZnO (5-50) μg/mL against isolates were also done by but less than our results (74).
Our results are in discrepancy with those reported by Daou et al. (2017), who demonstrated that TiO2-ZnO allowed complete eradication of C. albicans with very low MIC (75). On the other hand, the study found that the least inhibition zone of C. albicans was 8 mm in concentration 0.01 mg/ml of ZnO by Suha et al. (2015) in contrast to the results of Yousef et al. (2012) who showed that the best inhibition zone of C. albicans was 18 mm in concentration 10 µg/ml which is equal to 0.01 mg/ml the least concentration (76,77). Several studies have described the germicidal mechanisms of TiO2 nanoparticles involving the release of positively charged ions to reaction medium linked to (negative charges) thiol group (-S.H.) of the proteins on the cytoplasmic membrane (78). This response led to the imprisonment of the cell wall and augmented permeability. It deforms cellular components such as DNA, ribosomes, cellular enzymes, and microbial cell (79). So far, many studies have been conducted on the role of nanoparticles in the biological field (80)(81)(82)(83)(84)(85)(86)(87)(88). In this regard, it is best to study the relationship between the use of nanoparticles and some abnormalities and complications, such as genetic polymorphisms (89), mitochondrial diseases (90), sexual disorders (91) and overall human health (92) Impact.