Hydrothermal Assisted Biogenic Synthesis of Silver Nanoparticles And Their Anticandidal Activity On Virulent Candida Isolates From COVID-19 Patients

Fatma O Khalil National Liver Institute, Menou a University Enas M. Ghonaim National Liver Institute, Menou a University Shimaa Abed El-sattar National Liver Institute, Menou a University Sally W. Elkhadry National Liver Institute, Menou a University Hala El-Refai National Liver Institute, Menou a University Muhammad Babar Taj (  dr.taj@iub.edu.pk ) The Islamia University Bahawalpur Omar Makram Ali Turabah University College, Taif University Ahmed Salah A. Elgawad National Liver Institute, Menou a University Hospital Heba Alshater Menou a University Hospital, Menou a University


Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the primary infectious agent of Coronavirus Disease 2019 (COVID-19), a rapidly spreading pneumonia [1]. It is confronting the global public health system 2 . SARS-CoV-2 infects any age with a high impact on the elder people other than SARS-CoV and MERS-CoV 3 . Aside from the pathogenicity of SARS-CoV-2, microbial infections play a crucial role in the occurrence as well as the progression of SARS-CoV-2 infection by making a diagnosis, prognosis, and treatment of COVID-19 more di cult, as well as increasing illness symptoms and mortality 2 . Candida strains have been reported in the skin and mucosal dwellers that cause a variety of devastating diseases in immunosuppressed individuals and those who are susceptible 4 . Candida species possess numerous virulence factors, including exoenzymes like phospholipase as well as protease, or the capacity to generate the germ tubes and cling to buccal epithelial cells, that aid in the adherence as well as the intrusion of these organisms through the cell membrane, malfunctioning or rupturing 5 . The improper handling of antimicrobial drugs may enhance the prevalence of drug-resistant pathogens [2]. A major cause for the failure of antifungal treatment is low penetration potential to the infection site as well as their side effects [3].
Nanotechnology is one of the promising areas of science that has shown its impact on all scienti c elds equally [4,5]. Nanosized materials, having a size of 20-100 nm, have attracted the attention of researchers because of their propitious characteristics [6]. Engineered silver nanoparticles (AgNPs) have shown their outstanding applications in food-related elds owing to their remarkable antifungal activities [7,8] and are widely applicable in the biomedical eld like cancer photodynamic therapy, biomedical sensing, molecular imaging as well as drug delivery [9]. Synthesis of nanoparticles via green strategy such as by using plant extract [10], enzymes, phytochemicals etc are preferred mostly as compared to chemically manufactured nanosized material owing to overcome its toxic effect on the environment and living beings [11].
Thymus vulgaris L. plant has tremendous medical applications as it was used previously to treat various diseases like stiffening of arteries, urinary tract infections, dyspepsia, pneumonia, respiratory disorders (asthma, bronchitis, or cough), toothache, endocarditis [12][13][14] and septicaemia due to the presence of biochemical in it and it gains the status of the General Recognized as Safe (GRAS) de ned by Food and Drug Administration (FDA) [15].
In most antifungal treatments, the main biological resistance for drug diffusion is the formation of bio lm that is formed by aggregation of Candida albicans [19,20]. Drug diffusion decreases due to the formation of bio lm that enhances the cell viscosity [21,22]. The exact mechanism for antifungal treatment is still unknown for thyme coated silver nanoparticles. The present study aims to develop a low cost sustainable hrdrothermal method without addition of any base and with minimum invovement of energy to explore the application and effect of T/AgNPs on membrane damage against Candida isolates from Covid-19 patients.

Materials And Methods
This is a cross-sectional study done using fungal isolates from COVID-19 patients admitted in patients of Menou a Faculty of Medicine, Menou a University hospital after patients consent and approval of Research Ethics Committee of faculty of medicine, Menou a University (IRB No. 312021/FORE8). Fungal identi cation Antifungal testing and molecular detection of virulence factors were done in Microbiological laboratory, National liver Institute, Menou a University. All chemicals as well as solvents were of analytical grade and were not puri ed further.

Plant material
The Thymus vulgaris plant was collected from Egypt. The plant was distinguished at the o ce of Botany, Faculty of Science, El-Menou a University, Shebin El-Kom, Egypt where voucher examples were stored.

Preparation of plant extract
Fresh plant leaves were steeped in 70 percent ethanol/water (100 ml) at room temperature for 7 days after being cleaned with tap water and nally with deionized water. It was then ltered by utilizing Whatman lter paper no. 1 The extract was then warmed to 4 degrees Celsius and stored for future use.

Synthesis of silver nanoparticles
Thyme loaded silver nanoparticles were synthesized by modi ed hydrothermal method [23]. A 25 ml of 0.25 M silver nitrate solution was mixed with 25 ml of plant extract and stirred at 35 °C for 1 h. This solution mixture was transferred into Te on lined sealed stainless-steel autoclaves and kept in a hydrothermal oven at a temperature of 150 °C for 1.5 h. Then the contents allowed to cool to room temperature. Pure dark brown colored T/AgNPs were collected by centrifugation at 5000 rpm for 10 minutes. The T/AgNPs were dried in oven at 60 °C for 4 h, ground, and preserved in airtight bottle ( Figure   1).

Characterization of silver nanoparticles
The analysis was carried at National Research Centre Cairo Egypt. A Jasco dual-beam spectrophotometer was used to record the UV-vis spectroscopy estimates (model UV-VIS-NIR 570) operated at a resolution of 2 nm. FTIR measurements were recorded on Perkin Elmer Inc ranging from 4000 nm to 650 nm. Morphological ponders were employed utilizing high-resolution transmission electron microscopy (HRTEM). The HRTEM images were gotten by a JEOL-JEM-2100 version and an atomic force microscope (AFM) was used to determine surface topography and roughness pro les of Nanomaterials using Model 5600LS manufactured by Agilent technology company in the USA. The biological study was implemented in the National Liver Institute, Menou a University during the period from May 2020 to February 2021 on different samples isolated from COVID-19 patients. The samples were inoculated on Sabrouds dextrose agar( HiMedia, India) and colonies identi ed by Gram staining, morphology on cornmeal agar, germ tube tests, and chromogenic medium (HiMedia, Mumbai, India), and further identi cation and antifungal sensitivity were done by the VITEK-2 Yeast identi cation and AST card (BioMe ŕieux). microliters of 100 percent ethanol were poured over the lysate. After that, a centrifuge was used to separate the solution. The nucleic acid was extracted with 100 µl of the kit's elution buffer.

PCR ampli cation
Oligonucleotide Primer: The primers used were provided by Metabion (Germany) and are listed in (Table 1). A 25 µl reaction including 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer at 20 pmol concentration, 5.5 µl of water, and 5 µl of DNA template was used to test the primers. An Applied biosystems 2720 thermal cycler was used to carry out the reaction. The PCR products were eluted out using 5V/cm gradients on a 1.5 percent agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature. 15 µl of the products were inserted into each gel slot for analysis. The fragment sizes were determined using a general 100 bp ladder (Fermentas, Germany) as well as gelpilot 100 bp ladders. A gel documentation system (Alpha Innotech, Biometra) was used to photograph the gel, and the data was processed using computer software. (Table 3) lists the target genes, primer sequences cycle conditions and amplicon sizes, 2.5.4. Antifungal susceptibility testing The antifungal effect of biosynthetic T/AgNPs was evaluated on virulent Candida. albicans and non-albicans isolates using the disc diffusion method using discs; Nystatin 100 U, Clotrimazole 10µg, Fluconazole 10µL, Voriconazole 10µg, Itraconazole10µg, the results obtained were compared for T/AgNPs formed by using the chemical strategy and Thymus vulgaris. The disk diffusion method shows the magnitude of the susceptibility of the virulent fungi. The Minimal Inhibitory concentrations (MICs) for fungi were identi ed as the low quantity at which there is no visible growth can be seen.

Statistical Analysis
The mean and standard error were used to express all the data. To determine the relationship between qualitative variables, the Chi-square test will be performed. When more than 25% of the cells have an anticipated count of less than 5, the Fisher exact test will be applied for 2x2 qualitative variables.
When comparing the mean and SD of two sets of quantitative normally distributed data, the Student T-test will be employed, whereas the Mann Whitney test will be used when the data is not normally distributed. When comparing three or more groups with quantitative normally distributed data, the oneway analysis of variance (ANOVA) test will be performed, whereas the Kruskal-Wallis test will be used when the data is not normally distributed. When the P-value is less than 0.05, it is considered statistically signi cant.

UV-vis analysis
The hydrothermal synthesis of T/AgNPs using Thymus vulgaris plant extract was detected by visual inspection of the fabricated samples in addition, to monitoring the UV-vis spectra. Alteration of color to yellowish-brown in aqueous solution con rmed the formation of silver nanoparticles [27]. It was observed the color change of colorless solution of silver nitrate to yellowish-brown after 10 min of the addition of extract sample owing to the preparation of silver nanoparticles and this was evaluated by using UV-vis spectrophotometer as depicted in (Figure 2). At the end of the reaction, the solution of silver nanoparticles was investigated for stability. It was observed that nanoparticles were stable for a long time without being agglomerated. A small band at 438 nm was observed which appeared in the absorption spectra of the fabricated sample (0.1 ml sample). If the concentration of plant extract was increased, the band was positioned from 438 nm to 425 nm.
This band was identi ed to the absorption by colloidal silver nanoparticles in the visible region (380-450 nm) because of the excitation of surface plasmon vibrations [28]. The intensity of peaks increased and this is an indication of the increase of concentration of silver nanoparticles [29]. In addition, it was observed that the shifting of the band to lower wavelength from maximum absorption wavelength indicated the formation of smallsized Ag nanoparticles with an increased amount of Thymus vulgaris. So, it is manifested that there is a successful reduction to silver nanoparticles using Thymus vulgaris extract.

Fournier Infrared analysis (FTIR)
The identi cation of biomolecules that are responsible for the reduction and stabilization of silver nanoparticles were detected by FTIR. Thymus vulgaris has a heavy amount of protein and is highly concentrated in amino acids [30]. FTIR measurements for Thymus vulgaris extract and reduced silver nanoparticles were depicted in (Figure 3). FTIR transmittance peak at 3357 cm -1 was detected for Thymus vulgaris extract, which was allotted to OH stretching vibration, which become narrow and shifted to the high region at 3267 cm -1 . Other bands at 1700 and 1623 cm -1 were due to amide I and at 1443 cm _1 was assigned to amide II respectively and the peak at 1270cm -1 corresponds to amide III [31][32][33][34].
The modi cation in structure is an indication that the reduction, as well as stabilization of silver nanoparticles, advanced through the coordination between the nitrogen atom of the amide group as well as silver ions. It was shown by FTIR measurements that the amide group of protein has a greater capacity to bind metal. The proteins play a signi cant role as a capping agent as well as preventing the aggregation of nanoparticles.

High-resolution transmission electron microscopy (HRTEM) Analysis
TEM measurements are proceeded to evaluate the particle size as well as size distribution for the fabricated nanoparticles. (Figure 4). shows HRTEM images registered from the drop coated HRTEM grid of the manufactured Ag nanoparticles. Nanoparticles with agglomeration and spherical shape were depicted in micrographs which are of course due to the aggregation of silver nanoparticles. It is clear the presence of a gap in T/AgNPs shown in the HRTEM images due to covering of Thymus vulgaris extract and at lower concentration of Thymus vulgaris extract, fewer molecules were present which de cient in to cover the nanoparticle surfaces leading to the expedition of the aggregation. At some level of Thymus vulgaris extract content, all the nanoparticles are loaded, therefore stabilization occurs. So, Thymus vulgaris extract is utilized defensive agents to prohibit the aggregation through interaction with initial nanoparticles. and this is in a good agreement explanation of UV-vis spectroscopy results, which is distinguished from welldispersed silver nanoparticles. The small particle size of T/AgNPs is detected from 19 nm to 25 nm which is due to the enhanced amount of plant extract. Moreover, the size distribution becomes narrower as the amount of Thymus vulgaris plant extract exceeds. From these results, it is concluded that the size of the formed nanoparticles becomes smaller, and the particle size distribution is amended as the quantity of the extract rises.

Atomic force microscope (AFM) Analysis
The atomic force of microscopy was utilized for the determination of morphology and agglomeration of silver nanoparticles using Thymus vulgaris extract as shown in (Figure 5). Individual particles, as well as clusters of particles, can also be identi ed with the AFM. Microscope pictures are crucial in research and development initiatives, as well as when xing quality control problems. The AFM allows for three-dimensional viewing. The vertical, or Z, axis resolution is restricted by the instrument's vibration environment, whereas the horizontal, or X-Y, axis resolution is limited by the diameter of the scanning tip. Agglomeration of silver nanoparticles biosynthesized on the surface.

Antimicrobial activity
The antifungal treatment was performed by determining the clear inhibition zone supplied with 0.031-0.5mg/mL of silver nanoparticles. The development of the Candida population was observed to decrease by supplying 0.5 mg/mL of T/AgNPs ( Figure 6). The greater inhibition zone (22 mm) was monitored for Candida albicans, The MICs value observed for T/AgNPs from 0.125-0.250 mg/mL against Candida and bacteria species while MBCs and MFCs value obtained was 0.250 and 0.5000 mg/mL respectively. In a present research T/AgNPs with MIC values ranging from 156.25 to 1,250 µg/mL and MFC values ranging from 312.5 to 5,000 µg/mL against Candida kruzei, Candida glabrata and Candida albicans. It was indicated from MIC and MFC values that T/AgNPs has a greater potential for anticandidal activity. Moreover, Different amount of T/AgNPs ranging from 62.5 to 1000 µg/mL was also applied to check the effect on the development of Candida tropicalis Candida famata and Candida albicans species, followed by decreased growth of candida species at all concentration of the sample. The Candida species growth increases rapidly in the dearth of T/AgNPs. However, when the high quantity of T/AgNPs from 500 to 1000 µg/mL was applied, the Candida cells growth was ceased. It has been shown from ( Figure 6) that the growth inhibition of Candida cells was increased with an increased amount of T/AgNPs. The Minimal Inhibitory concentrations (MICs) for fungi were identi ed as the lowest quantity at which no visual growth was observed. The Minimum fungicidal concentration (MFC) [35,36] was de ned as the lowest drug concentration at which subcultures yielded negative ndings or less than three colonies, indicating that >99% of the original inoculum had died.

Morphological and ultrastructural alteration caused by T/AgNPs
The structural and morphological modi cations due to T/AgNPs on Candida albicans were detected by TEM. It was detected that when Candida albicans cells were subjected to 50 and 100 µg/mL T/AgNPs, a remarkable change in the cell wall and cell membrane was observed ( Figure 6). It was found from TEM analysis that T/AgNPs were adhered not only on the surface of the cell wall and membrane but also penetrate the cell and stored in the cytoplasm followed by the rupturing of the cell wall and destruction of the cytoplasmic membrane ( Figures 6). The recent TEM analysis is in good agreement with the previous work on morphological analysis of the effects of T/AgNPs on Candida albicans. The exact Anticandidal mechanism of nanostructured material is not known. Kim et al studied that the cell membrane and cell wall of the Candida albicans species were destroyed by T/AgNPs due to the formation of "pits and holes" on the surface which prevents the process of budding and results in cell death. Gutierrez et al. [37] reported his work on antifungal activity of T/AgNPs due to inhibition of β-glucan synthase and structural modi cations of cell wall which loss its proper functioning and followed by cell death. Furthermore, in other literature, it was reported that T/AgNPs enhance apoptosis in mitochondria, DNA, and nuclear fragmentation, phosphatidylserine externalization as well as the activation of metacaspases in Candida albicans and leads to cell destruction due to the build-up of intercellular ROS. Radhakrishnan et al studied that Candida albicans cell destruction is not only due to the accumulation of ROS but also the T/AgNPs affect the cellular microenvironment membrane uidity, structure, and ultrastructure, cellular ergosterol levels, as well as fatty acid composition, particularly oleic acid, which is essential for hyphal morphogenesis Candida albicans isolate resistant to antifungal agents; Nystatin 100µg, miconazole (10 µg), Clotrimazole (50µg), ucytosine (25µg) and sensitive to Fluconazole(25µg). taking subcultures on the middle plate showing that the Candida isolates in the are resistant to Thymus vulgaris alone (lower part) and sensitive to silver nitrate alone; the zone of inhibition increases on the addition of biologically active silver nitrate solution in the sabrouds agar plate on the right.

High-resolution Transmission Electron Microscopy (HRTEM)
To visualize the action of biologically active silver nitrate on Candida albicans isolates (Figures 8, 9,10 & 11) After growth, the fungal cells were centrifuged as well as xed with 2.5% glutaraldehyde as well as 4% paraformaldehyde (Sigma-Aldrich) and nally contrasted in the solution of 5% uranyl acetate. Dehydration was performed using acetone (Sigma-Aldrich). The samples were analyzed under a transmission electron microscope (Thermo Fisher Scienti c, USA).
The sociodemographic features, clinical and laboratory data and prognosis of the studied patients with and without fungal growth are mentioned in (Table 2). Candida albicans isolates features as mentioned in (Table 3) regarding antifungal sensitivity and virulence factors. (Figures 12 and 13) show the effect of Thymus vulgaris alone and after union with silver nitrate particles on Candida albicans isolates in comparison to other antifungal agents. Fisher=14.55 0.002** Candida species, Aspergillus, and P. jirovecii infections were found in critically unwell COVID-19 patients. parenteral feeding, broad-spectrum antibacterial medication, mechanical ventilation, indwelling central venous as well as bladder catheters, lymphopenia, comorbidities, older age, and corticosteroids 14 are all prevalent in COVID-19 patients admitted to the ICU. The goal of this study was to characterise fungal infection in COVID-19 patients while also investigating the antifungal effects of silver nitrate biosynthesized by Thymus vulgaris.

Discussion
The hydrothermal method has been adopted for the biogenic synthesis of silver nanoparticles. The main advantage of this method is that it can produce a bulk amount of silver nanoparticles and the e ciency of this procedure is very much high as compared to other known methods. The synthesized T/AgNPs have been well characterized by various techniques. To check the anticandidal activity, forty-six fungal isolates were obtained from different specimens of 60 COVID-19 inpatients; 33 were Candida albicans, 10 Candida nonalbicans isolates (2 Candida famata, 4 Candida galabrata, 2 Candida krusei, and 2 Candida tropicalis). It has been reported that bacteria species and fungi are considered co-infections in critically ill patients with COVID-19, which increases its morbidity and mortality15. Numerous strains of Candida also have been identi ed as unique pathogens from COVID-19 patients' pulmonary specimens (TA, BAL as well as BAS) and recognized as a source of co-infections [38] and the frequency of fungal co-infection was signi cantly greater than previously reported [38].
It is found that Candida albicans isolates were highly virulent as 90.7% of isolates had ALS3 and 76.7% had the Hyr1 gene. Hyr1 and Als3 are hyphaspeci c genes and the hyphal regulator RAS1 is related to adhesion and increase fungal pathogenicity [39]. The RAS1 was prevalent in 88.4%. Ras signalling is crucial to the integration of environmental cues with morphogenesis and virulence [40].  [44]. Fluconazole treatment was discovered to be a potential risk for gene mutation and overexposure, which leads to long term uconazole-resistant C. parapsilosis [45]. Plant extracts, which contain biomolecules and plant metabolites including avonoids, tannins, and terpenoids, polyphenols as well as algae and fungal metabolites, have special qualities that allow them to be employed as a reducing and capping agent for the manufacture of pharmaceuticals and stabilisation of nanoparticles (NPs) in a variety of elds, including biomedical, pharmaceutical, and food industries [46]. In the current study, Thymus vulgaris derived silver nanoparticles showed a signi cant antifungal effect with a decrease in the number of resistant isolates than known antifungal uconazole, miconazole, nystatin, clotrimazole and ucytosine (P-value <0.05). This nding was in line with Mohammadi, M., et al., 2019, who found that Ag NPs manufactured from thyme extract have no cytotoxicity at concentrations below 3.5 ppm, making them a viable alternative to Fluconazole for treating super cial fungal infections [47]. Similar results were obtained by Anjugam, M., et al, 2018 [48]. The excellent use of plant mediated silver nanoparticles in antibacterial as well as anticancer activities has led to a surge in strong interest among different research groups to employ it, reported by Fahimirad, S.,et al, 2018 [49].

Conclusion
In our study silver nanoparticles were biosynthesized by an eco-benign, clean, and useful approach, using leave extract of Thymus vulgaris. UV-visible spectroscopy, FT-IR, AFM, and TEM techniques were utilized to characterize synthesized silver nanoparticles. The UV-Visible spectroscopic results revealed that the NPs synthesized from leave extract of Thymus vulgaris lies within the range of 19-25 nm and the which was con rmed with the published literature. The morphology and agglomeration of silver nanoparticles using Thymus vulgaris extract were done by atomic force microscopy. Moreover, synthesized nanoparticles have a great antifungal effect. It was concluded that AgNps formed by medicinal plant extracts have remarkable antifungal activity and can be used in various antimicrobial drugs. The authors declare no con ict of interest to publish the paper.

Funding
This study has been funded by the Taif University Researchers Supporting Project number (TURSP-2020/81),         The percentage of resistant isolates to chemical antifungal agents and T/AgNPs Figure 13 Anti-fungal sensitivity testing of commercial antifungal versus biogenic silver nitrate