Treatment isolated fungi from laboratory tools in some Baghdad hospitals by using biosynthesized nanoparticles

The study aims to biosynthesized of sliver nanoparticle from aqueous extract of olive leave and evaluate the effectiveness of the synthesis AgNPs against isolated fungi. The study mediating fifty samples were taken from various tools in laboratory from five hospitals in Baghdad. Four species of fungi were identified depending on the morphological and microscopic characteristics. The most common isolated fungi based on their frequency ratio were as follows Aspergillus niger 87.5%, Aspergillus flavus 62.5%, Aspergillus fumigatus 53.5% and Aspergillus nidulans 37.7%.The Biosynthesis of silver nanoparticle developed a rapid, eco-friendly and convenient green method for the stable silver nanoparticles (AgNPs) were synthesised with an average diameter of 30 ± 60 nm and like spherical in shape, using the aqueous solution of the Olive tree ( Olea europaea ) leaves extract.The reaction is carried out at 10 -3 M of silver nitrate. The AgNPs synthesized were confirmed by their change of color to (dark brown-grey). The characterization was studied using UV-Visible spectroscopy, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Inhibition effect of AgNPs against fungi has been studied using well diffusion method by studying the effect of different concentration (100, 75, 50 and 25). The results revealed that the AgNPs have considerable antifungal activity comparison with alcohol. The obtained results indicate that the highest level of inhibition zone was detected at the concentration of 100 µg/ml of AgNPs, where the inhibition zones are (23.33 ± 4.41) for A. flavus and the lowest level of inhibition zone was detected at the concentration 25 µg/ml of AgNPs ,where the inhibition zones are (6.00 ± 1.15) for A.nidalus . While using alcohol the highest level of inhibition zone was detected at the concentration of 100 µg/ml of Alcohol, where the inhibition zones are (12.33 ± 1.45) for A.nidalus , and the lowest level of inhibition zone was detected at the concentration 25 µg/ml of Alcohol ,where the inhibition zones are (4.67± 0.33) for A.flavus.


Introduction
Fungal contamination in health care facility had been the subject of many studies. These have observed that high percentages of hospital infections are caused by fungi, such as Candida spp and various species of Aspergillus, Mucor, and Cladosporium [1]. Apparently, Different fungi are causing infection; some of them are usually occurring infections while the others are rare [2]. Aspergillus is widely distributed in nature and found throughout the world, it seems to adapt to a wide range of environmental conditions and heat resistant conidia provide a good mechanism for dispersal. Disinfection process is removing pathogenic (kill or inhibiting growth) from materials or tools and prevent disease transmission, a process is less perfective than sterilization, which is Physics or chemical process include removal all microorganism, including spores (bacteria, fungi and virus) [3]. Nanoparticles exhibit completely new or improved properties based on specific characteristics such as size, distribution and morphology. Nanoparticles present a higher surface to volume ratio with decreasing size of nanoparticles. Specific surface area is relevant for catalytic reactivity and other related properties such as antifungal activity. Olive leaves extract (OLE) are rich in biophenols (BPs), such as oleuropein (Ole) which is the major active components in leaves , and its derivatives, verbascoside, ligstroside, tyrosol or hydroxytyrosol , as well as cafeic acid , p-coumaric acid , vanillic acid, vanillin, leteolin, diosmetin, rutin , luteolin-7-glucoside , apigenin-7glucoside and diosmetin-7glucoside [4].These compounds with antifungal activity can be explored and used for the control of fungal diseases. However, the olive leaves extract and AgNo 3 lead to the formation of Silver nanoparticles (AgNPs) were used to inhibit or killing of pathogenic fungi. Many surveys have displayed the upshot effect of nanoparticles against some of the pathogenic fungi [5].The study aimed to biosynthesized of sliver nanoparticle from aqueous extract of olive leave and evaluate the effectiveness of the synthesis AgNPs against isolated fungi and comparison the effect with the effectiveness of Alcohol.

Materials and Methods Samples collection:
From November in 2017 to February 2018, fifty samples were taken using sterile transport media swabs from various tools in the laboratory such as (loop, hood, incubator, refrigerator, cork borer, slide, and water bath.) from five hospitals in Baghdad.

Identification of fungi:
Different colonies of fungi were observed on (SDA) at 37°C for 5-10 day and appeared with different characteristic features such as (Colonies dense, Colonies dark green,Colonies compact white or yellow Colonies at first white to pale yellowish, Creamish-yellow in daylight, more grayish in darkness ,Light grayish ,Colony pale brownish-gray.Colony smooth radial furrows, whitish to creamcolored or grayish-brown). Through the duration of incubation, different fungal colonies were subjected to microscopic and macroscopic to note their growth, mycelium nature and structure of hyphae. The growth of Filamentous fungal as mold and yeast on SDA, were sub cultured on separate SDA culture dishes. The incubation had been at 37°±1 C for one plate, while the others at 25°±1 C. The growth of the Pure culture for each yeast and mold colonies were examined to show their microscopic structures under magnification and clarified using mycological keys manuals.
Microscopic examination was made by the examination of many preparations from different areas of fungal growth mounted on a clean slide with lactophenol cotton blue stain to reveal spores which include large septate macroconidia and small, single-celled microconidia. The slide was gently heated in a spirit lamp in order to facilitate the staining and remove air bubbles (the excess stain was removed by a tissue paper) and then the cover slip was applied [7].

Aqueous extracts preparation:
Olive leaves were collected from olive trees in Baghdad University, Baghdad, Iraq. The leaves were washed for several clock time with distilled water to remove the particles of dust, then dried to remove the residual moisture and stinger it into small pieces. A measure of 100g from the small pieces of olive leaves was placed into the flask with 500ml of sterile distilled water and heated in the water bath at 60 o C for 2hr. Then the extract was cooled to room temperature and filtered for several times with Whitman no.1 filter paper by Buchner funnel. The aqueous solution of the extract was concentrated by using a rotary evaporator to remove the largest possible amount of water, and then put it in Petri dish at room temperature to dry. The stock solution of the extract was prepared with a concentration of 1 and1.5 mg/ml [9].

Biosynthesis of silver nanoparticles:
An aqueous solution (1mM) of silver nitrate (AgNO3) were prepared and used for the synthesis of AgNPs. 5 ml of plant extract added into 95 ml of a solution of 1mM silver nitrate for reduction into Ag+ ions. In a typical synthesis of silver (Ag) nanoparticles, the leaf extract (1.5 ml) was added to 30 ml of 10 -3 M AgNO3 aqueous solution in a 250 ml Erlenmeyer flask and heated on a water bath at 75 °C for 60 min. Reduction of silver nitrate to silver ions was confirmed by the color change from colorless to brown .The formation of AgNPs confirmed by spectrophotometry determination. The fully reduced solution centrifuged at 5000 rpm for 30 min. The supernatant liquid discarded and the pellet obtained re-dispersed in deionized water. The centrifugation process repeated two to three times to wash off any absorbed substances on the surface of the AgNPs [10].

Characterization of AgNPs: Ultraviolet-visible spectroscopy:
An Ultraviolet-Visible spectrophotometer (UV-Vis) refers to absorption spectroscopy. The samples were measured by UV-VIS double beam spectrophotometers from 300-600 Wave length.

Atomic force microscopy (AFM)
The surface morphology of the nanoparticles was visualized by Atomic force microscope (Veeco) under normal atmospheric conditions. The examined samples were dispersed on a small slide and explored on contact mode of the instrument [11].

Scanning electron microscopy (SEM)
The morphological characterization of the samples was done using JEOL Jsm6480 LV for SEM analysis. The samples were dispersed on a slide and then coated with platinum in an auto fine coater, after that the material was subjected to analysis [12].

Estimation of antifungal activity of silver nanoparticle: Inoculum preparation
For the susceptibility testing of filamentous fungi, the inoculum prepared by serial dilutions method of the pathogenic fungi suspension Agar Well Diffusion Method: The different concentrations of silver nanoparticle (100, 75, 50 and25) mg/ml and suspension (1ml) of 10 -1 and 10 -3 dilution of pathogenic filamentous fungi were homogeneously mixed with SDA medium using the pour plate method in 6 cm Petri dishes. Then a well of (5mm) was made in the medium by using sterile cork borer. 100µl of each concentration of the silver nanoparticle was transferred into separate wells. Plates are incubated at 35±2°C for 1 day in Aspergillus spp. The diameter of the inhibition zone was recorded for each replicate and the average diameter was calculated [13].

Statistical Analysis
The Statistical Analysis System- [14] program was used to show effect of different factors in study parameters least significant difference -LSD test was used to significant compare between means in this study.

RESULTS and DISCUSSION Indicator of Fungal Occurrence and Frequency:
In Tables-(1 -4) showed that the highest occurrence and frequency of fungi is Aspergillus niger (96%) and (87.5%) respectively. This may be attributed to its high ability to produce enzymes and secondary metabolites which enable to exploit various minerals sources and resist wide ranges of environmental disinfectants [15].This result agreed with [16] showed that Aspergillus niger had highest occurrence and frequency of fungi .

Synthesis and Characterization of Silver Nanoparticles: 1-Visual Observation and UV-Vis Spectral Study:
After the first detection of silver nanoparticles presence in the chemical reaction vessel after the incubation periods which was the changeover of the reaction mixture from colorless to pale yellow, which indicated initial reduction, then to brownish yellow to light brown to deep brown -grey-with the time. UV-vies spectrophotometer was the next step to characterize the biosynthesized AgNPs. UV-Visible spectroscopy was used to examine the size and configuration of nanoparticles in aqueous suspension. Formation and stability of prepared AgNPs in sterile distilled water were approved by UV-Vis spectrophotometer in a range of 300 -800 nm of wavelength. Once olive leaves extract was mixed with AgNO3, the reduction response of Ag+ ion to Ag o was observed by measuring the UV-Vis spectrum for the reaction media. (Figure-1) showed the recorded UV-Vis spectra after the completion of the reaction.

Characterization by Scanning Electron Microscopy (SEM):
The scanning electron microscope was employed to analyze the shape of the silver nanoparticles that were synthesized by a green method. The surface-deposited silver nanoparticles are clearly seen at high magnification (100,000 kx) in the micrograph. SEM analysis shows that the olive leaf plant has tremendous capability to synthesize silver nanoparticles which were predominantly spherical in shape and were uniformly distributed with an average size little less than 50 nm (Figure-2).

Characterization by Atomic Force Microscopy (AFM)
Atomic force microscopy was used as a confirmatory technique to characterize the biosynthesis of AgNPs by detection their average diameter in addition to morphology in both two dimensions and three dimensions. The results obtained in this study showed that the biosynthesized AgNPs by olive leaf plant have average diameter 50.28 nm as shown in Table-3 and Figure-(3 a, b and c).   Table-4 showed the inhibition zone (mm) of fungal growth after treatment with AgNPs at the concentration 100,75 , 50and 25 µg/ml. The results showed that the maximum level of inhibition zone at (23.33 ± 4.41) for A flavus when the concentration of 100 µg/ml of AgNPs. While, the minimum level of inhibition zone at (6.00 ± 1.15( for A.nidalus when the concentration 52 µg/ml of AgNPs.. It was noted that the inhibition level increase with the increasing of AgNPs concentration at all different cases. Alike studied by [17], they investagated a high reduction of fungal growth due to the higher AgNPs concentration. The inhibitory effect of AgNPs on the growth of fungi caused by [18]:  Liberation of lipopolysaccharides.  Changing of permeability of cell tissue layer.

Antifungal activity of Alcohol (Ethyl alcohol):
The obtained results indicate that the highest level of inhibition zone was detected at the concentration of 100 µg/ml of Alcohol, where the inhibition zones are (12.33 ± 1.45) for A.nidalus, and the lowest level of inhibition zone was detected at the concentration 25µg/ml of Alcohol ,where the inhibition zones are (4.67 ± 0.33) for A. flavus. The results were summarized i Table-5. Ethanol is well known as an inhibitor of the growth of microorganism. It has been reported to damage mitochondrial DNA in yeast cells [19], and to cause inactivation of some enzyme, such as hexokinase and dehydrogenase .Nevertheless, some strains of the Fungi appearance tolerance and can adapt to high concentrations of ethanol [20].Cell tissue layer has received extensive consideration as primary targets of ethanol stress. Many reports have suggested a relationship between the fatty acid physical composition of lipid membranes and ethanol stress tolerance [21]. This effect can be obtained by adding directly ethanol to the surfaces but at present, there is no available information about modeling the effect of ethanol on fungal outgrowth [22].

Conclusion
Many fungi isolated from laboratory tools, this result indicated the contamination in laboratory tools, Biosynthesized silver nanoparticles from aqueous extract of olive. Nanoparticles, aqueous extract of olive and alcohol used as antifungal, Silver nanoparticles more effective than aqueous extract of olive and alcohol that uses in sterilizing.