1. INTRODUCTION:

Nanotechnology is a technology that has attracted great interest in recent years and deals with nanometer sized items¹. Nanoparticles with one or more dimension s(≥100 nm), have appealed attention due to their unique properties and applications^2,3, they present in nature e.g. DNA is 2.5nm⁴,a virus is ~100 nm⁵and a bacterium ~1-3μm⁶. The chemical composition, size, and shape of nanoparticles influence their properties^7,8. Physical, chemical, and biological methods synthesize diverse types of nanoparticles^9,10, although these methods are more popular, their biomedical applications are limited due to the toxic chemicals used¹¹. Therefore, the need to develop a nontoxic, safe clean and eco-friendly method for the preparation of nanoparticles became necessary ¹².

One of these methods is to use microorganisms to synthesize nanoparticles. The processing conditions for nanoparticle biosynthesis were reduced. Allowing the synthesis at physiological pH, temperature, pressure, and at a minor cost. A huge number of microorganisms have been found to synthesize metallic nanoparticles, either intra or extracellularly^13-15. Many bacteria and fungi have revealed the ability to biosynthesize inorganic and metal nanoparticles with properties similar to that of chemically-synthesized materials and all have their own advantages and disadvantages. Prokaryote has received the most attention in this field due to their ability to adapt to extreme conditions, they are also easy to cultivate and manipulate. In commercial biotechnological processes e.g. bioleaching and bioremediation, the microorganisms can accumulate metals and the growth conditions can be easily controlled and the interaction between microorganisms and metals has been previously discussed^16,17.

Microorganisms have adapted the high levels of metal by mechanisms that altered the toxic metal chemical nature so that it no longer sources toxicity, resulting in the formation of nanoparticles of the metal.

Bacteria have different mechanisms for the synthesis of metal NPs, these include detoxification/reduction of the metal to less toxic metal salts and decreases in the membrane permeability through cationic membrane transport systems that transport important cations for metabolism¹⁸. Mechanisms elucidation were identified through metal-resistant bacterial strains mutagenesis and subsequent complementation of mutants to identify resistant genes¹⁹. Magnesium oxide is currently used as an antacid for heartburn and sour stomach, MgO NP has been used as adsorbents and catalysts^20,21 and as an antibacterial agent against S. aureus and E. coli by damaging the cell wall of bacteria or production of reactive oxygen species (ROS), also have acted as the antibiofilm formation of common pathogens^22,23. These nanoparticles approved their antibacterial effect against both Gram positive and Gram negative organisms which is dependent on both the particle size and concentration. These nanoparticles are safe for humans and the environment and can be used in grouping with other antibacterial agents^. MgO NP also showed promising results by the removal of both chemical and biological substances in water purification^24-25.

Statistical optimization of nanoparticles production is the primary task in a biological process²⁶. Seven variables affecting the production process have been optimized using the first order model Placket –Burman design to evaluate the optimal levels of the significant variables. The statistical method used to investigate multiple process variables with fewer experimental trials under different conditions²⁷.

The objective of this study was the environmental friendly biosynthesis method of metallic MgO NP also to identify significant variables influencing their production using Plackett–Burman design, the antimicrobial and ant biofilm activities of MgO NP were also studied.

2. MATERIALS AND METHODS:

2.1 Chemicals:

MgSO4.7H2O, Nutrient broth media, isopropanol, and polyvinylpyrrolidone(PVP) were obtained from Sigma–Aldrich (St Louis, MO, USA). Other media components used were of the highest purity and obtained from (Oxoid) and (Difco).

2.2 Radiation Source:

Gamma irradiation was applied at NCRRT, Cairo, Egypt. using Cobalt 60 sources (Gamma cell 4000-A-India). The applied dose rate was fixed at 2.02 KGy/h. Gamma rays produce a free radical and solvated electrons after water radiolysis.

2.3 Isolation and screening of bacteria for MgO NP synthesis:

Samples of soil were collected from the Ismailia canal and sewage. Enrichment culture technique was applied²⁸. Nutrient agar medium was used to isolate bacterial strains. One gram of soil samples was serially diluted to 10-4in distilled water, shacked and cultured in Nutrient agar medium containing fluconazole 150 mg as an antifungal. The plates were incubated at 30°C for24 hours then stored at 4°C with monthly subculture maintenance. The promising isolated strain was identified using Polymerase chain reaction 16s rDNA and electrophoresis analysis and registered in Gene bank with accession no MT102429.

2.4 Molecular identification:

2.4. 1DNA extraction:

Genomic DNA of the bacterial isolates was extracted using the Gene JET Genomic DNA purification kit 98 (Thermo Scientific # k0721).

2.5 PCR amplification and sequencing of 16S rRNA gene:

The 16s rRNA fragments were partially amplified by PCR using Maxima Hot Start PCR Master Mix (Thermo K1051) in Sigma Company of Scientific Services, Egypt (www.sigma-co-eg.com). For phylogenetic analysis, the determined sequences were compared with the sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov) by BLAST search.

2.6 MgO NP biosynthesis:

After bacterial isolation and purification, each sterile bacterial isolate was cultured separately in 250ml Erlenmeyer flask containing 100ml nutrient broth. The Erlenmeyer flasks were kept in a shaker at 30°C for 48 hours after that the cultures were centrifuged by cooling centrifuge at 4°C and 10,000 rpm, the supernatant (cytoplasmic fraction) was taken and saved for the synthesis of MgO NP.

20ml of the supernatant were mixed with 20 ml of 4mM MgSO4, 0.2% isopropanol, and 0.2% P.V.P (Polyvinylpyrrolidone). In case of radiation (then subjected to a dose of 15 and 25kGy of gamma radiation). The formation of MgO NP was confirmed by visual observation of brownish color. Then further confirmed by UV– Visible spectrophotometer at a range of 200–800 nm²⁹. Cell filtrate used as control under the same experimental conditions.

2.7 Preparation of solid powder sample:

For pellet preparation, 35ml of MgO nanoparticles solution was taken in falcon tubes and centrifuged at 10000rpm for 15 min. Pellet was thoroughly washed 3 times with double distilled H₂O and centrifuged for 10 min at the same speed. Pellet was dried in a hot air oven at 40°C and the pure powder obtained was used for XRD, TEM, FTIR analysis, and consequent studies.

2.8 Characterization of MgO NPs:

Characterization of MgO NPs was done by UV–Visible spectrophotometer (JASCO V-560. UV–Vis. Spectrophotometer) to analyze the absorption patterns at (300-800 nm), using the negative control for the auto-zero. The size and morphology of the synthesized MgO NPs(were recorded using TEM (JEOL electron microscopy JEM-100 CX). FT-IR measurements were carried out to obtain information about chemical groups present around MgO NPs. Finally, X-ray Diffraction study was completed to investigate the MgO NPscrystallinity, using X-ray diffraction patterns-XRD 6000 series, Shimadzu, Tokyo, Japan.

2.9 Plackett Burman design was used to optimize biosynthesis of Mg ONP

A screening experiment in which the most important factors concerning the synthesis of MgO NP were elucidated by applying Plackett and Burman design,³⁰ This design is recommended when more than five factors are under investigation³¹. Seven factors (Salt concentration(mM), filtrate concentration(ml), PVP (%), isopropanol (%), time (hr), temperature (°C) and radiation (KGy) in nine combinations organized according to the design. All trials were performed in duplicates and the dry weight average of the synthesized MgO NP was treated as a response. The main effect of each variable was simply calculated as the difference between the average of measurements made at the high setting (+) and the average of measurements made at the low setting (-) of that factor. t-values and p-value were also calculated for the determination of variable significance. The significance of the model was determined by ANOVA, the regression equation was obtained, values which represented the level of P value< 0.05 were considered to be significant.

2.10 Antibacterial activity of Mg ONP:

2.10.1 Invitro antimicrobial bioassay:

Antibacterial activity of MgO NP was assessed against the Gram-negative bacteria (Escherichia coli NRRL-B210) and gram bacteria (Staphylococcus aureus ATCC25923) by well diffusion agar method³². The optical density of the inoculum was adjusted at 0.125 for freshly grown bacteria, wells were made to add MgO NP (100mg/ml), and ceftriaxone was used as an antimicrobial reference for bacteria. The plates were incubated for 24 h at 37°C. Antimicrobial activities were evaluated by measuring the inhibition zone diameter (cm).

2.11 Minimum inhibitory concentration (MIC):

The MgO NP powder was dehydrated and sterilized in an oven at 200°C for at least one hour before each in vitro experiment. Various concentrations (10, 30, 40 and 70 mg/ml) of MgO NP were prepared and added into series of test tubes containing 5ml of sterilized nutrient broth and 0.1ml of E.coli NRRL-B210 as gram –ve and Staphylococcus aureus ATCC25923 as gram +ve culture and allowed to grow at 35^oC for 24h., bacteria alone with the nutrient broth was kept as control and they were examined for inhibition studies. MIC was the lowest concentration of the nanoparticle that did not permit any visible growth of bacteria. CFU was calculated by sub-culturing the above (MIC) serial dilutions after 24 h in nutrient agar Petri plates using 0.01ml loop and incubated at 35^oC for 24h ³³.

2.11.1 Investigate the effects of MgO NP against biofilm:

2.11.2 A qualitative assessment:

Biofilm formation was determined as described by ³⁴. 0.5mL Brain Heart Infusion (BHI) Broth media with 2% sucrose and 0.5ml MgO NP (sub MIC) was inoculated with a loopful of bacteria from overnight culture plates. On the other hand, BHI media with 2% sucrose was inoculated with a loopful of test bacteria (as acontrol) and incubated for 24 hours at 35°C. The tubes were decanted and washed with distilled water, driedand stained with crystal violet (0.1%). Excess stain was removed and tubes were washed with deionized water and dried in an inverted position to be observed for biofilm formation. The amount of biofilm formation was scoredas non-biofilm, moderate, or high biofilm ³⁵.

2.11.3 Tissue culture plate method (TCM):

The anti-adhesive activity of MgO NP was studied against clinical strains of Gram-positive and Gram-negative (E.coli NRRL-B210 and Staphylococcus aureus ATCC25923) using micro-titration plate methods ³⁵. The bacterial suspension (100 μl) in brain heart infusion broth with 2% sucrose was added with sub MIC(60mg)nanoparticles together in the same well for each isolate. The next well filled with (100μl) brain heart infusion broth with 2% sucrose and (20 μl) bacterial growth suspension without nanoparticles (as control), the plate was incubated at 35°C for 24h. Non adherent cells were removed by gently washing twice the wells with sterile distilled water plates, dried at room temperature then stained with 200 μl crystal violet solution for 20 min, the excess stain was rinsed off by thorough washing with deionized water and plates were kept for drying for 15 min at room temperature then 200 μl ethanol (95%) added for each well and read using microElisaauto reader. Biofilm development was assessed by measuring the optical density (absorbance at 630nm) using a spectrophotometer.

3. RESULTS AND DISCUSSION:

3.1 Isolation and Screening of Bacterial Isolates for MgO NP Synthesis:

All bacterial isolates were inoculated on nutrient broth and incubated for 2 days at 35°C to examine the production of MgO NP. The optical absorption spectra of MgO NP were recorded using UV–Vis Spectrophotometer. The width of the peak and absorbance of wavelength (data not shown) indicated that from all the nine isolates. Bacillus paramycoides strain MCCC 1A04098 it was registered in Gene bank with accession no MT102429. The isolate was the best to synthesize MgO NP. A strong UV-Vis absorption peak at 300 nm indicated the formation of MgO NP and broadening of peak indicated that the particles are polydispersed. The absorption peak width of the surface Plasmon depends on the shape and size of the metal nanoparticles ³⁶.

3.2 Identification and phylogenetic analysis:

The promising isolated strain was determined by the 16 s rRNA gene sequence (1000 bp). The results indicated that the isolated strain 16 s rRNA gene sequence was similar to that of many species of the genus Bacillus spp. According to the GenBank database and a BLAST search ^37-38. The results in Figure 1 showed that the isolate was found to be closely related to the Bacillus paramycoides strain MCCC 1A04098 with 98.75% identity. (Figure-1)

3.3 Statistically optimization of MgO NP synthesis by Placket Burman design:

MgO NP synthesized by different microorganisms are greatly influenced by the reaction conditions³⁹.The upper and lower limits for the parameters were fixed on the basis of preliminary experiments. A nine-run table was generated by Placket Burman statistical design, Table 1 showed the experimental design with seven variables (X1: salt concentration (mM), X2: filtrate concentration (ml), X3: PVP (%), X4: isopropanol (%), X5: time(hr), X6: temperature(°C) and X7: radiation(KGy )affecting MgO NP biosynthesis, each variable was tested in 3 levels and showed a variation in both dry weight (0.1-3.0 mg/50 ml) and particle size (15.5 to 35mm).

The main effect for each variable (data not shown) for affecting both MgO NP dry weight (yield) and particle size were investigated, the reaction temperature (°C), PVP (%) and filtrate concentration (ml) had a positive effect on MgO NP synthesis while the other variables had a negative effect, thus investigate the optimum conditions that have the most significant effect on the dry mass weight and the particle size of the produced MgO NP.

ANOVA analysis was summarized in Table 2 R² of the model is 99%. The predicted R² (0.99) is in acceptable agreement with the adjusted R2 (0.99) as shown by the actual predicted plot, the P-value of the model is significant (0.0207). In order to determine the accuracy of this experiment, a verification experiment was carried out in a triplicate (data not shown).

The average particle diameter for MgO NP was 15.5 to 35 nm. The results showed that in trials 1and 2 the particle size ranged between 30-35 nm, this may be contributed to the effect of both low filtrate and PVP conc., while the temp was at a high level. Trials 7, 8, 9 particle size was less than 20 nm, with high production yield for 7, 8 (3, 2 mg/50 ml respectively). The nanoparticles sizes may also affect the antimicrobial activity, the smaller silver nanoparticles exhibited higher efficiencies against pathogens, and thus, the size of MgO NP may also impact the antimicrobial activities ⁴⁰.

Table1 Optimization of MgO NP production by placket-Burman design.

Trial No.	Variables							Dry weight (mg)/ 50 ml	Average of particle size (nm)
Trial No.	X1	X2	X3	X4	X5	X6	X7	Dry weight (mg)/ 50 ml	Average of particle size (nm)
1	-(2)	-(0.5)	-(0.1)	+(0.3)	+(96)	+(100)	-(non)	0. 2	35
2	+(6)	-(0.5)	-(0.1)	-(0.1)	-(48)	+(100)	+(25)	0. 3	30
3	-(2)	+(1.5)	-(0.1)	-(0.1)	-(48)	-(37)	+(25)	0.4	22
4	+(6)	+(1.5)	-(0.1)	+ (0.3)	+(96)	-(37)	-(non)	0.1	20
5	-(2)	-(0.5)	+ (0.3)	+ (0.3)	-(48)	-(37)	-(non)	0.2	20
6	+(6)	-(0.5)	+ (0.3)	-(0.1)	+(96)	-(37)	-(non)	0.4	20
7	+(6)	+(1.5)	+ (0.3)	-(0.1)	-(48)	+(100)	-(non)	3.0	15.5
8	+(6)	+(1.5)	+ (0.3)	+ (0.3)	+(96)	+(100)	+(25)	2.0	17.5
9	0(4)	0(1)	0 (0.2)	0 (0.2)	0(48)	0(50)	0(15)	0.45	18

Table 2 ANOVA analysis for MgO NP synthesis.

Term	cooficient	Std Error	t –value	p-value
Intercept	35.45234	1.151985	30.78	0.0207*
Salt concentration (mM)	0.012815	0.011877	1.08	0.4758
Filtrate concentration(ml)	2.595349	0.051423	50.47	0.0126*
PVP(%)	5.440699	0.102845	52.9	0.0120*
Isopropanol(%)	-2.4744	0.273191	-9.06	0.07
Time(h)	-0.0445	0.003185	-13.98	0.0454*
Temperature(°C )	0.0185	0.000323	57.27	0.0111*
Radiation(K Gray)	-0.007	0.000545	-12.96	0.0490*

Fig.4 X-ray Diffraction analysis of MgO NPs.

3.4.2 FT-IR analysis of nanoparticles:

FT-IR analysis confirm the presence or absence of certain functional groups, to determine the formation of nanoparticles. The FT-IR spectrum of MgO NP synthesized in T7 Figure 3 showed vibrational-absorption with strong and better resolved bands of 3445.39, 2077.72, 1634.71, 1114.72 and 676.18cm^-1respectively, indicating the presence of N–H stretching of the secondary amide of the protein, the stretching vibrations of C C or O–H bending mode, C–N stretching vibrations of aromatic amines, the stretching variation of C–O–C and MgO NP respectively, this result were Inco ordinance with EL-Moslamyet al⁴⁶.

El-Batal et al²⁹ reported that 3445.39 cm-1 was corresponding to the vibrational stretching O–H bond of the carboxyl acid this agree with our results.

3.4.3XRD analysis of MgO NP:

XRD was used for detection of the crystallinity of MgO NP, it elucidates the status of the atoms, axes, and size. The data of the MgO NP XRD was shown in Figure4. Diffraction characteristics are displayed inside 2ɵ (degree) as 28.3, 31.7, 44.5, 55.4, 65.9and, 76 where some peaks represent the Bragg’s appearances (0 0 1), (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) extensions in that position sequentially, that can be verified to the levels of cubic MgO (JCPDS 75)⁴². MgO NP was a crystal that produced the face-centered cubic (fcc) crystalline configuration, this agreed results with El-Sayyad et al³⁹. The standard peaks for MgO were detected in the XRD spectrum, signifying that the expected crystalline form of MgO was present. Additional peaks in the XRD pattern of the MgO sample also designated the presence of Mg (OH)₂ phase. MgO is hygroscopic and can eagerly react with water in the atmosphere to form Mg(OH)₂ that explained the presence of Mg(OH)₂ peaks in the XRD spectrum.

3.4.4 TEM Analysis of the Nanoparticles:

TEM was used to characterize the synthesized MgO NP Figure 5. The results confirmed that MgO NP had rounded shape and size ranging from 7.81 nm to 20.67 nm in trial 7 and the mean of average of size was 15.5 MgO NP size and shape was uniform in trial 7.

Fig. 5 TEM image of MgO NPs.

3.5 Antibacterial activity:

MgO NP prevent the growth of microorganism by an electrochemical model of action to penetrate and disrupt their cell walls, leakage of metabolites occurs and other cell functions are stopped, thereby inhibiting the organism from functioning or reproducing⁴⁷.

The antibacterial activity was tested using both G +ve bacteria (S.aureus ATCC25923) and G −ve (E. coli NRRL-B210). The antibacterial effect of MgO NP has the lowest production cost related to silver and titanium dioxide and nanoparticles⁴⁸. The minimal inhibitory concentration (MIC) of any antibioticsis the minimum concentration necessary to inhibit the visible growth of a microorganism in culture. The MgO NP (MIC) was determined in a range 10, 30, 40 and 70 mg/ml concentration (data not shown). The results in Figure 6. showed that MgO NP at 70 mg/ml concentration have antibacterial activity against the G +ve bacterium (S. aureus) and G −ve bacterium (E. coli), approved by an inhibition zone 3.5cm for G +ve and 2.2 cm for−ve bacterium). Vidic et al ²⁵ reported that MgO NP has antibacterial activity against both Gram positive and Gram-negative bacteria which depended on the particle size and concentration. This result agreed with, Imani and Safaei²³, they found that magnesium oxide nanoparticles have antibacterial activity against Gram-positive bacteria rather than Gram-negative bacteria. Many reports have shown that MgO NP has better activity towards gram-positive bacteria than towards gram negative bacteria due to the difference in the structure of the cell membrane ⁴⁹. Therefore, E. coli shows a stronger resistance to MgO NP, compared to S. aureus ^50.

Fig.6 Antibacterial activity of MgO NP : (A) E. coli NRRL-B210, (B) S.aureus ATCC25923

3.6 Effect of MgO NP on biofilm formation:

Microorganisms producing biofilm considered the main virulence factor causing biofilm-related infections. Bacteria embedded in biofilm (sessile form) are more resistant to antimicrobials than planktonic bacteria, about 65%–80% infections are caused by biofilm forming microbes ⁵¹. Our study Figure7(A and B)

Showed that the addition of MgO NP (60mg/mL) concentration into the culture for 24h. MgO NP represented the highest anti-adhesive percentage against S.aureus ATCC25923 (90%) and E. coli NRRL-B210 (93%).

Fig.7 Effect of MgO NP on biofilm formation using (A) tissue culture plate method, (B) Qualitative assessment.

4. ACKNOWLEDGMENTS:

The authors would like to thank the National Research Center, Department of Chemistry of Natural and Microbial Products for financing and supporting this study.

5. CONFLICT OF INTEREST::

Conflict of interest: The authors declare that they have no conflict of interest.

6. ETHICAL STATEMENT:

This article does not contain any studies with human participants or animals performed by any of the authors.

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Received on 07.01.2021 Modified on 22.04.2021

Research J. Pharm. and Tech 2022; 15(1):63-70.

DOI: 10.52711/0974-360X.2022.00012