Antimicrobial and Antibiofilm Potency of Green Silver Nanoparticles Synthesized by Aqueous Garlic Extract Against Microbes Causing Otitis Media CURRENT STATUS: UNDER REVIEW

Aim of study: Otitis media (OM) is highly prevalent worldwide and is the main cause of hearing loss in developing countries. Resistant microorganisms and biofilm formation have become a major health and economic problem. The aim of this study was to establish an eco-friendly method for synthesis of silver nanoparticles using aqueous garlic extract and sunlight. Moreover, detect its potential to inhibit otitis media microbes. Methods: Basically, highly resistant and biofilm forming otitis media pathogenic isolates were determined by Kirby-Bauer disc diffusion method and their biofilm production qualitatively was tested. Then, compared their susceptibility to tetracyclin 30µg (for bacteria) and nystatin 100 µg (for fungi) alone and with different concentrations of green synthesized silver nanoparticles (AgNPs), as well as, their biofilm production. AgNPs were prepared by easy, fast, non-toxic, economically eco-friendly way, using the direct sunlight irradiation on aqueous garlic extract. Their characterization by UV- visible spectral analysis, TEM, DLS, XRD and FTIR were studied. Results: Two multidrug resistant bacterial isolates; Pseudomonas aeruginosa and Bacillus cereus were detected as biofilm producers with black colonies on congo-red agar and three resistant fungal isolates to nystatin; Penicillium chrysogenum, Aspergillus niger and Aspergillus flavus were tested against green synthesized AgNPs. Highly significant (p-value<0.001) antimicrobial and antibiofilm activity of biosynthesized AgNPs against tested pathogens were illustrated in this study. TEM images for Pseudomonas aeruginosa and Aspergillus flavus after treated with AgNPs, were exhibited shrinkage in the cytoplasmic materials, rupture of cell walls,

3 precipitation of AgNPs particles inside the cells and biofilm was mostly disseminated.
Conclusion: aqueous garlic extract that used to stabilize the AgNPs may be ideal natural candidates for future studies exploring their safely use in biomedical applications especially against the repeated infections caused by multi drug resistant (MDR) and biofilm forming microbials as in otitis media. introduction Otitis media (OM) resulted from combination of different pus-forming microorganisms (viruses, bacteria and fungi), coming from the nose and nasopharynx by way of the eustachian tube and aggregate at the mucous membrane lining the middle ear cleft leading to inflammation in this membrane and effusion of fluids into the middle ear due to infection (1). The extraordinary occurrence of OM in developing countries population but with high prevalence in children (second most common infection in children less than 2 years) (2) due to immaturities in their immune system (3). Chronic otitis media affect language development. In severe cases, it may lead to hearing loss (2). Moreover, it is the most common reason for antibiotic and antifungal consumption because it is well-known reappearance infection and the antibiotics enduring to be the basis for treating OM with its complications (4-6).
Unregulated and unnecessary use of antibiotics resulted in a constant decrease in their effectiveness, leading to the development of multi-drug-resistant (MDR) bacterial strains. For that reason, it is essential to search for alternative healthcare approaches to diminish the problem of bacterial infections and contaminations (7).
Several antimicrobial agents such as amoxicillin, cefotaxime, cefoxitin, tobramycin, 4 gentamicin, tetracycline, chloramphenicol, ciprofloxacin, ofloxacin, ampicillin and erythromycin are mainly prescribed for the treatment of bacterial otitis media (8)(9)(10)(11). Prevention of the OM is very important as the WHO has reported that hearing impairment in 42 million people (above 3 years) in the world was mainly caused by OM (2, 12).
Garlic (Allium sativum) is a very popular and famous plant from ancients till days all over the world. In addition, aqueous garlic extract (AGE) offers extra benefits of fabrications of highly stable, biocompatible and spherical nanoparticles with proteins and amino acids as a natural capping an stabilizing agents (13).
Silver nanoparticles (AgNPs) have global attention due to their unique properties and their numerous applications especially in biomedical such as antimicrobial ointment, medical diagnostics, biological sensing, biological control, cancer therapy, drug delivery…etc (14).
AgNPs have low toxicity to human cells, effective broad-spectrum activity against bacteria, and a lower probability to cause microorganisms resistance than conventional antibiotics (15). The generation of new efficient antimicrobial resulted from known week antibiotics with a very low concentration of AgNPs offers an advanced perspective in the medical field. However, it diminishes the long-stay costs and the side-effects of antimicrobial therapy and surgery (6).
Synthesis AgNPs by chemical and physical routs have drawbacks making them unsuitable for biomedical applications. Meanwhile, green synthesis of AgNPs (using plant, enzymes or microorganism) is rapid, ease in size control, energy-efficient, facile non-toxic, environmentally conscious and friendly. In addition, to these benefits, use of plant extract for AgNPs synthesis offers a wide range of benefits over other biological synthesis methods because it is available, does not require the 5 maintenance of cell cultures, safe in handling, and incorporates support for the large-scale synthesis of nanoparticles, presence of many metabolites act as reducing or capping/stabilizing agents (13, 16).
This study aims to synthesize the AgNPs by easy, fast and economically eco-friendly route via the aqueous extract of garlic cloves and use a very small amount of resulted AgNPs to study their influence on the susceptibility of pathogenic microorganisms isolated from OM.

Collection of Samples
All the isolates were obtained from the culture collection at Drug Microbiology Lab, Drug Radiation Research Department at (NCRRT), Cairo, Egypt. Garlic (Allium sativum) were purchased from local markets in Cairo, Egypt.
All the bacterial isolates were re-cultured on MacConkey and nutrient agar media. Antimicrobial discs were applied to the surface of the inoculated agar plate with sterile forceps and gently pressed down onto agar surface.The plates were incubated for 3-5 days at room temperature (22-25°C). The sizes of the zones of inhibition interpreted by referring to standardized chart.
Detection of biofilm production for highest resistant bacteria using Congored detection method (18) described a simple qualitative method to detect biofilm production by using congo red agar (CRA) medium. Thirty-seven grams of brain heart infusion broth powder, 50g of sucrose and 15g of agar was dissolved in one liter of distilled water then autoclaved at 121°C for 15 minutes for sterilization. Eight grams of Congo red indicator powder was dissolved in one liter of distilled water then autoclaved at 121°C for 15 minutes. Then it was added to the autoclaved brain heart infusion agar with sucrose at 55°C. plates were inoculated and incubated aerobically at 37°C for 24 hours.
Isolates that produced black colonies with dry crystalline consistency were regarded as biofilm positive, while those showing pink colonies were biofilm negative. The experiment was performed in triplicate.

Green synthesis of silver nanoparticles using aqueous garlic (Allium sativum) extract based on sunlight irradiation (19)
For the reduction of silver ions, 25 ml of aqueous garlic extract (AGE) was added to 475 ml of 0.1 M AgNOȝ (8.5g AgNOȝ + 475ml deionized water) to give a stock solution of colloidal silver nanoparticles (AgNPs) with concentration of 100µg/ml. This reaction mixture was stirred properly and exposed to bright sunlight. Within

Fourier Transform Infra-Red Spectroscopy (FTIR):
FT-IR investigation was a helpful method that provides information regarding chemical functional groups remaining in the AGE. The measures were carried out following a JASCO FT-IR 3600 Infrared spectrometer by working KBr Pellet purpose.
It was recorded at a resolution of 4.0cm -1 in a wave number range of 400-4000 cm -

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Antimicrobial activity of green synthesized AgNPs compared with tetracycline, nystatin and AGE against selected isolates.
Two fold serial dilutions of colloidal AgNPs were prepared from stock concentration (100µg/ml) using deionized water. Fresh bacterial suspension were adjusted to 0.5 McFarland's standard and swabbed over MHA. Sterilized Whattman no.1 filter papers, 6 mm in diameter were loaded with 10 µl of (100, 50, 25, 12.5 and 6.25 µg/ml) of synthesized colloidal AgNPs, 500mg/ml of aqueous garlic extract (AGE) also was loaded. In addition, disc of tetracycline 30 µg (TE) were used for bacterial isolates and nystatin 100 µg/m for fungal isolates to compare their influence on the sensitivity of the tested isolates. All plates were incubated at 37°C for 24 hours, 9 followed by the measurement of the diameters of the inhibition zones.

Morphology and biofilm production of bacterial and fungal isolates before and after treatment using (TEM):
In order to obtain an electron micrograph of a bacterial and fungal specimen, the later must be subjected to a series steps, including fixation, dehydration embedding, sectioning and staining. Ultrathin sections were picked up on carbon grids and viewed on (JEOL-JEM 1010) Transmission Electron Microscope, Japan.
Located at the Regional Centre for Mycology and Biotechnology-Al-Azhar University, Cairo, Egypt.

Statistical analysis
Recorded data were analyzed using the statistical package for social sciences, version 20.0 (SPSS Inc., Chicago, Illinois, USA). Quantitative data were expressed as mean± standard deviation (SD). Qualitative data were expressed as frequency and percentage. Different statistical steps were done. First, ANOVA used when comparing between more than two means. Then, LSD was used for multiple comparisons between different variables. Finally, the confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the p-value was considered highly significant (<0.001), significant (<0.05) and insignificant (>0.05).

Susceptibility test
Disc diffusion test was performed for 80 bacterial and fungal isolates from otitis media. The percent were 59% and 41%, respectively. Out of them, six types of bacteria and three fungal isolates were selected as the highly resistant isolates.
Bacteria were tested against 9 antibiotics and fungi (Penicillium chrysogenum, Aspergillus niger and Aspergillus flavus) were considered as resistant if inhibition zone around nystatin disc is less than 13 mm.

Biofilm (slime) production by Congo red agar
Biofilm production in bacterial pathogens were determined qualitatively by Congo red agar method (CRA). Only 2 bacterial isolates (Bacillus cereus and Pseudomonas aeruginosa) gave black colored colonies on CRA as a positive indication for slime production in these isolates (Figure 2).

Synthesis and Characterization of silver nanoparticles (AgNPs)
Synthesis of AgNPs were confirmed initially by changing the colorless suspension of AgNo3 with AGE into brown after exposed to irradiated sunlight ( Figure 1).
The Fourier transform infra-red spectroscopy (FTIR) spectra were used to characterize the functional groups and the molecular interaction between the synthesized AgNPs and AGE. As shown in (Figure 7) and (Table 1) Antimicrobial activity of AgNPs were tested against two biofilm forming highly resistant bacteria (Bacillus cereus and Pseudomonas aeruginosa) in comparison with AGE and tetracycline as control where bacteria were resistant to it. The different concentrations of AgNPs had an increased inhibition zones than AGE and tetracycline, this was considered a statistically highly significant difference where p-value<0.001 as shown in (Figure 8).

Transmission electron microscopy (TEM) for ultrastructure integrity of biofilm forming bacteria (Pseudomonas aeruginosa):
The isolated Pseudomonas aeruginosa, which is considered as a highly pathogenic bacteria, was treated with AgNPs.
The ultrastructure study showed the cells of the untreated Pseudomonas aeruginosa, where the protoplast can be differentiated into a cytoplasmic region, which is rich in ribonucleic acid, distributed throughout the whole cytoplasm, and chromatic region contain some ribonucleic acid of the cell. The cell wall looks rigid, intact and retaining its structural integrity. The robust biofilm was clearly illustrated as shown in Figure 9 (A) with different magnification power. Meanwhile, Figure 9  who reported the presence of polydispersed and spherical nanoparticles of varying sizes.
It was observed that the particle size obtained from DLS measurement was obviously larger than TEM results, because DLS measures the hydrodynamic radius which take into consideration the native protein on the surface of AgNPs as well.
The XRD outline obviously displayed that the silver nanoparticles formed by the In the present study, colloidal AgNPs at higher concentrations had an increased inhibition zones against the tested fungal isolates than nystatin and AGE. These 16 results was in harmony with (31) who found that photosynthesized AgNPs exhibited higher antifungal activity.
The ultrastructure study using the transmission electron microscope was carried out for the microbial isolate Pseudomons aeruginosa treated with aqueous garlic extract (AGE). The results in this study were in consistence with ( declarations -Ethics approval and consent to participate: Not applicable.
-Consent for publication: Not applicable.
-Availability of data and material: Please contact author for data requests. -Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
-Authors' contributions:  Figure 1 Green synthesis of silver nanoparticles (AgNPs). AgNO3 suspension and aqueous garlic extra  FTIR spectra of AGE-AgNPs and AGE alone. TEM studies section of Aspergillus flavus, (A) a typical untreated hypha was with normal cell