EVALUATION BIOSYNTHESIZED SILVER
NANOPARTICLES BY PHOMATROPICA
AGAINST SOME MULTIDRUG RESISTANCE
BACTERIAL ISOLATES
Thaer Ali Hussein
Department of Biology, College of Education, Quran, University of Basrah, Basrah,
Iraq
thbasra2@gmail.com
Ismail J. Abbass
Department of Biology, College of Education, Quran, University of Basrah, Basrah,
Iraq
Afrodet A. Salah
Department of Pathological Analysis, College of Science, University of Basrah,
Basrah, Iraq
Reception: 10/11/2022 Acceptance: 08/01/2023 Publication: 29/01/2023
Suggested citation:
A. H., Thaer, J. A., Ismail and A. S., Afrodet. (2023). Evaluation Biosynthesized
Silver Nanoparticles By Phomatropica Against Some Multidrug Resistance
Bacterial Isolates. 3C Tecnología. Glosas de innovación aplicada a la pyme, 12
(1), 296-319. https://doi.org/10.17993/3ctecno.2023.v12n1e43.296-319
https://doi.org/10.17993/3ctecno.2023.v12n1e43.296-319
3C Tecnología. Glosas de innovación aplicadas a la pyme. ISSN: 2254-4143
Ed.43 | Iss.12 | N.1 January - March 2023
296
EVALUATION BIOSYNTHESIZED SILVER
NANOPARTICLES BY PHOMATROPICA
AGAINST SOME MULTIDRUG RESISTANCE
BACTERIAL ISOLATES
Thaer Ali Hussein
Department of Biology, College of Education, Quran, University of Basrah, Basrah,
Iraq
thbasra2@gmail.com
Ismail J. Abbass
Department of Biology, College of Education, Quran, University of Basrah, Basrah,
Iraq
Afrodet A. Salah
Department of Pathological Analysis, College of Science, University of Basrah,
Basrah, Iraq
Reception: 10/11/2022 Acceptance: 08/01/2023 Publication: 29/01/2023
Suggested citation:
A. H., Thaer, J. A., Ismail and A. S., Afrodet. (2023). Evaluation Biosynthesized
Silver Nanoparticles By Phomatropica Against Some Multidrug Resistance
Bacterial Isolates. 3C Tecnología. Glosas de innovación aplicada a la pyme, 12
(1), 296-319. https://doi.org/10.17993/3ctecno.2023.v12n1e43.296-319
https://doi.org/10.17993/3ctecno.2023.v12n1e43.296-319
ABSTRACT
Researchers describe the extracellular manufacture of silver nanoparticles (AgNPs)
from Phomatropica and its effectiveness against certain multidrug-resistant
pathogenic bacteria that were obtained from the Central Laboratory of Quran Hospital.
These bacteria were pathogenic.
The AgNPs were synthesized and characterized by scan electrons microscopy,
Fourier transform infrared spectroscopy, UV-visiblespectrophotometer that established
the mostly spherical nanoparticles synthesis with size range between 55-99 nm. The
potential antimicrobial activity was reported vs (Staphylococcus aureus,
Pseudomonas aeruginosa, Klebsiella pneumonia, and Escherichia coli)by well
diffusion method.AgNPs showed different inhibitory areas at different concentrations,
the 50µg/mlconcentration of AgNPsappeared inhibition zones varied from(0-21 mm),
while at 100 µ
g/mlofAgNPs varied between (13-25mm) vs the tested pathogenic
bacterial strains in this investigation. Nevertheless, the synergetic impact of AgNPs
with antibiotics have beendetected in the increasing the inhibitory impact vs the
pathogenic bacteria.
In conclusion, Extracellular biosynthesis appears to be a scalable and sustainable
process. Because of their biogenic nature, these Ag-NPs might be a better medication
candidate and have the potential to completely eliminate the issue of chemical
agents.Antibiotic-resistant bacteria are proliferating at an alarming rate. To address
this issue, the development of bactericidal agents is critical. AgNPs may provide a
solution for drug-resistant bacteria.
KEYWORDS
Phomatropica; Extracellular; AgNPs ; Biosynthesized ;Multidrug resistant MDR
PAPER INDEX
ABSTRACT
KEYWORDS
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. MATERIALS
2.2. ISOLATION AND IDENTIFICATION OF FUNGUS
2.3. COLONY CHARACTERIZATION
2.3.1. MORPHOLOGICAL AND MICROSCOPIC VIEW OF FUNGUS
2.3.2. MOLECULAR IDENTIFICATION
2.3.2.1.DNA EXTRACTION
2.3.2.2.POLYMERASE CHAIN REACTION (PCR) USING UNIVERSAL
PRIMERS
2.3.2.3.NCBI BLAST
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2.4. PREPARATION OF BIOMASS
2.5. FUNGAL MEDIATED SYNTHESIS OF AGNPS
2.6. SILVER NANOPARTICLE DETECTION AND CHARACTERIZATION
2.6.1. SONICATION
2.6.2. ASSAY USING UV-VISIBLE SPECTROPHOTOMETRY
2.6.3. FTIR (FOURIER TRANSFORM INFRARED SPECTROSCOPY)
ANALYSIS
2.6.4. SCAN ELECTRONS MICROSCOPE (SEM)
2.7. ANTIBACTERIALACTIVITY OF AGNPS
2.8. ASSAY FOR DETERMINING SYNERGISTIC EFFECTS.
2.9. EVALUATION OF THE INCREASE IN FOLD AREA
2.10.STATISTICAL ANALYSIS
3. RESULTS
3.1. FUNGAL IDENTIFICATION
3.2. GENETIC IDENTIFICATION OF PHOMATROPICA
3.2.1. GENOMIC DNA EXTRACTION
3.2.2. PCR AMPLIFICATION
3.2.3. SEQUENCING OF ITS GENE
3.3. SILVER NANOPARTICLE (AGNP) BIOSYNTHESIS
3.4. CHARACTERIZATION OFBIOSYNTHESISAGNPS
3.4.1. SONICATION OF AGNPS SOLUTION
3.4.2. UV-VISIBLE SPECTROPHOTOMETRY ANALYSIS
3.4.3. SCAN ELECTRONS MICROSCOPE (SEM)
3.4.4. FTIR ANALYSIS
3.5. AGNPS HAVE ANTIBACTERIAL ACTIVITIES
3.6. COMBINATION EFFICACY OF AGNPS WITH AMPICILLINAND
CHLORAMPHENICOL
4. DISCUSSION
4.1. ISOLATION AND IDENTIFICATION OFPHOMATROPICAFROM SOIL
4.2. BIOSYNTHESIS AND CHARACTERIZATIONOF AGNPS
4.3. UV-VISIBLE SPECTROPHOTOMETRY ANALYSIS
4.4. BIOSYNTHESIZED DESCRIPTION AGNPS BY SCAN ELECTRONS
MICROSCOPY (SEM)
4.5. FT-IR ANALYSISA OF THE BIOSYNTHESIZED AGNPS
4.6. AGNPSANTIBACTERIAL ACTIVITY ON PATHOGENIC BACTERIA
4.7. COMBINATION EFFICACY OF AGNPS WITH AMPICILLIN AND
CHLORAMPHENICOL
5. CONCLUSION
6. ACKNOWLEDGMENT
REFERENCES
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2.4. PREPARATION OF BIOMASS
2.5. FUNGAL MEDIATED SYNTHESIS OF AGNPS
2.6. SILVER NANOPARTICLE DETECTION AND CHARACTERIZATION
2.6.1. SONICATION
2.6.2. ASSAY USING UV-VISIBLE SPECTROPHOTOMETRY
2.6.3. FTIR (FOURIER TRANSFORM INFRARED SPECTROSCOPY)
ANALYSIS
2.6.4. SCAN ELECTRONS MICROSCOPE (SEM)
2.7. ANTIBACTERIALACTIVITY OF AGNPS
2.8. ASSAY FOR DETERMINING SYNERGISTIC EFFECTS.
2.9. EVALUATION OF THE INCREASE IN FOLD AREA
2.10.STATISTICAL ANALYSIS
3. RESULTS
3.1. FUNGAL IDENTIFICATION
3.2. GENETIC IDENTIFICATION OF PHOMATROPICA
3.2.1. GENOMIC DNA EXTRACTION
3.2.2. PCR AMPLIFICATION
3.2.3. SEQUENCING OF ITS GENE
3.3. SILVER NANOPARTICLE (AGNP) BIOSYNTHESIS
3.4. CHARACTERIZATION OFBIOSYNTHESISAGNPS
3.4.1. SONICATION OF AGNPS SOLUTION
3.4.2. UV-VISIBLE SPECTROPHOTOMETRY ANALYSIS
3.4.3. SCAN ELECTRONS MICROSCOPE (SEM)
3.4.4. FTIR ANALYSIS
3.5. AGNPS HAVE ANTIBACTERIAL ACTIVITIES
3.6. COMBINATION EFFICACY OF AGNPS WITH AMPICILLINAND
CHLORAMPHENICOL
4. DISCUSSION
4.1. ISOLATION AND IDENTIFICATION OFPHOMATROPICAFROM SOIL
4.2. BIOSYNTHESIS AND CHARACTERIZATIONOF AGNPS
4.3. UV-VISIBLE SPECTROPHOTOMETRY ANALYSIS
4.4. BIOSYNTHESIZED DESCRIPTION AGNPS BY SCAN ELECTRONS
MICROSCOPY (SEM)
4.5. FT-IR ANALYSISA OF THE BIOSYNTHESIZED AGNPS
4.6. AGNPSANTIBACTERIAL ACTIVITY ON PATHOGENIC BACTERIA
4.7. COMBINATION EFFICACY OF AGNPS WITH AMPICILLIN AND
CHLORAMPHENICOL
5. CONCLUSION
6. ACKNOWLEDGMENT
REFERENCES
https://doi.org/10.17993/3ctecno.2023.v12n1e43.296-319
1. INTRODUCTION
Nanotechnology, which deals with particles on the microscopic scale ranging in size
from 1 to 100 nanometers, is one of the most active study disciplines in current
material research (Saif et al., 2016). AgNPs stand out among the many forms of
metallic nanoparticles due to the broad-spectrum antibacterial effects they might
produce (Prabhu and Poulose, 2012; Rai et al., 2014; Gupta et al., 2017; Loo et al.,
2018). These nanoparticles are able to attach themselves to the membranes of
bacteria as well as the cell walls, and they might even penetrate the cells themselves.
They cause disruptions in the pathways that are used for signal transduction, they
cause damage to cellular structures, and they create reactive oxygen species (Kim et
al.,2011; Dakal et al., 2016) In the sectors of health and agriculture, AgNPs were
utilized to suppress hazardous microbes (Kim et al., 2012; Mishra and Singh, 2015;
Burdusel et al., 2018).
The vast majority of the currently available methods for producing nanoparticles
have downsides, including the usage of potentially harmful chemicals and the
production of waste that is detrimental to the environment (Iravani et al., 2014; Ahmed
et al., 2016). Because of this, there was a significant increase in interest in methods of
synthesis that are less harmful to the environment in the most recent few years.
Microorganisms that are capable of degrading metal salts and producing
nanoparticles of the required size and shape are used in the methods. These
microbes include bacteria, fungus, and plants (Azmath et al., 2016)
An alternative that is safe, non-toxic, and beneficial to the environment is the
biological reduction of metals, which results in the production of nanoparticles (Banu
and Balasubramanian, 2014). Since they possess a high tolerance for metallic and
are simple to handle, fungi are promising agents for the biogenic production of AgNPs
(AgNPs). They also create a significant number of extracellular proteins, which
contribute to the nanoparticles' already impressive level of stability (Balaji et al., 2009;
Du et al., 2015; Netala et al., 2016). In comparison to bacterial cultures, fungal
cultures provide a number of benefits, including a higher rate of biomass production
and the elimination of the need for additional steps to obtain the filtrate (Gade et al.,
2008).
2. MATERIALS AND METHODS
2.1. MATERIALS
Potato dextrose and potato dextrose agar (PDA) have been achieved from
( Himedia, India ) the antibiotics ( Chloramphenicol30mg) was obtained from
( Himedia, India ), (Ampicillin 10mg ) was obtained from (Rosteo, Italy ). and silver
nitrate (AgNO3) have beenbought fromSigma-Aldrich (Germany) and lactophenol
cotton blue from Merke ( India ).
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2.2. ISOLATION AND IDENTIFICATION OF FUNGUS
Phomatropica was isolated from soil sample collected different location in Basrah
(Southern Iraq) during the year 2022. Soil specimens was taken from approximately
(2-5) cm depth.To isolate soil fungus, the serial dilution method was used. To obtain
concentrations between one and four, a one-gram soil specimens has been
consecutively diluted in sterilized purified water (10-1 to 10-4). Each dilution was
transferred aseptically in 0.1 ml increments onto PDA plates. To distribute the sample
evenly, a sterilized glass spreader was used. At pH 6.0 and 28 °C, the plates were
cultured for 5-6 days. The fungal isolates are sub-cultured on (PDA) plates to produce
pure culture. Pure-isolates are kept in a refrigerator at 4 ºC for future research. The
isolated fungus was identified using visual characteristics, microscopic structure, and
molecular identification.
2.3. COLONY CHARACTERIZATION
2.3.1. MORPHOLOGICAL AND MICROSCOPIC VIEW OF
FUNGUS
Phomatropica which is used in the biosynthesis of AgNPs, was isolated from soil
and kept alive by maintaining it on PDA medium at 28 degree centigrade and 4
degree centigrade. The colony morphology and micro morphology of the fungus,
including the( color, shape, texture of the mycelia, spore formation pattern, etc), were
used to identify it. The fungus was also cultured on a PDA medium at 28 degree
centigrade for 10 days to analyze its colony morphology. Slide culture was used to
investigate the fungus's micro morphological traits. Cultures were grown on PDA
slides and cultured there for five days at 28 degree. The slides were then dyed with
lactophenol cotton blue and investigated under a microscope light (Dongyanget
al,2021). The taxonomic description led to the identification of the isolated fungus (R.
Schneid. & Boerema,1975 ;Vaibhar, 2012).
2.3.2. MOLECULAR IDENTIFICATION
2.3.2.1.DNA EXTRACTION
The extraction of DNA from fungi was performed using the method descrybed by
(Alshehri and Palanisamy,2020) and following the protocol instructions included in the
kit (Presto™ Mini gDNA Yeast Kit/ Genaid/ USA).
2.3.2.2.POLYMERASE CHAIN REACTION (PCR) USING
UNIVERSAL PRIMERS
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2.2. ISOLATION AND IDENTIFICATION OF FUNGUS
Phomatropica was isolated from soil sample collected different location in Basrah
(Southern Iraq) during the year 2022. Soil specimens was taken from approximately
(2-5) cm depth.To isolate soil fungus, the serial dilution method was used. To obtain
concentrations between one and four, a one-gram soil specimens has been
consecutively diluted in sterilized purified water (10-1 to 10-4). Each dilution was
transferred aseptically in 0.1 ml increments onto PDA plates. To distribute the sample
evenly, a sterilized glass spreader was used. At pH 6.0 and 28 °C, the plates were
cultured for 5-6 days. The fungal isolates are sub-cultured on (PDA) plates to produce
pure culture. Pure-isolates are kept in a refrigerator at 4 ºC for future research. The
isolated fungus was identified using visual characteristics, microscopic structure, and
molecular identification.
2.3. COLONY CHARACTERIZATION
2.3.1. MORPHOLOGICAL AND MICROSCOPIC VIEW OF
FUNGUS
Phomatropica which is used in the biosynthesis of AgNPs, was isolated from soil
and kept alive by maintaining it on PDA medium at 28 degree centigrade and 4
degree centigrade. The colony morphology and micro morphology of the fungus,
including the( color, shape, texture of the mycelia, spore formation pattern, etc), were
used to identify it. The fungus was also cultured on a PDA medium at 28 degree
centigrade for 10 days to analyze its colony morphology. Slide culture was used to
investigate the fungus's micro morphological traits. Cultures were grown on PDA
slides and cultured there for five days at 28 degree. The slides were then dyed with
lactophenol cotton blue and investigated under a microscope light (Dongyanget
al,2021). The taxonomic description led to the identification of the isolated fungus (R.
Schneid. & Boerema,1975 ;Vaibhar, 2012).
2.3.2. MOLECULAR IDENTIFICATION
2.3.2.1.DNA EXTRACTION
The extraction of DNA from fungi was performed using the method descrybed by
(Alshehri and Palanisamy,2020) and following the protocol instructions included in the
kit (Presto™ Mini gDNA Yeast Kit/ Genaid/ USA).
2.3.2.2.POLYMERASE CHAIN REACTION (PCR) USING
UNIVERSAL PRIMERS
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The fungus's internal transcribed spacer region was amplified using PCR.
(Schochet al, 2012).the ITS region of 5.8S rDNA genewas amplified by PCR reaction,
using universal forward and reverse
Primer ITS1-F :5’-TCCGTAGGTGAACCTGCGG-3’
and
Primer ITS4- R :5’- TCCTCCGCTTATTGATATGC-3’
(Raja et al, 2017).
2.3.2.3.NCBI BLAST
Basic Local Alignment search tool (BLAST) and National Center for Biotechnology
Information (NCBI) both provided insurance for Phomatropica.
2.4. PREPARATION OF BIOMASS
For the manufacture of AgNPs, the Phomatropica was the organism of choice. 250
gm of potato and 20 gm of dextrose are to be added for every liter of purified water. To
eliminate the medium component from the mycelia biomass, the mycelia were filtered
out of the culture broth using Whatman filter paper No. 1 and washed three times in
sterile Milli-Q deionized water. This process was done to remove the medium
component. Ten days were spent incubating Erlenmeyer flasks on a rotary shaker at a
temp of 25 degree centigrade and 120 revolutions per minute. At a temp of 25 degree
centigrade, a 250 ml Erlenmeyer flask was agitated continuously for three days using
the same method while it contained 10 g of biomass (wet weight) and 100 ml of
deionized water. After incubation, the fungal cell filtrate (FCF) was collected by filtering
the solution using Whatman filter paper and a Millipore filter with a pore size of 0.45
microns. Filtrate has beenclam and used in order to bring about the desired results of
producing AgNPs.
2.5. FUNGAL MEDIATED SYNTHESIS OF AGNPS
For the creation of AgNPs, 100 ml of fungal cell filtrate (FCF) was mixed with 0.017
gm of AgNO3 (Sigma-Aldrich 99.9%, Germany) to achieve a final amount of 1 mM,
and the mixture was then left to sit at 28°C in the dark for 72 hours. As a control, flaks
with FCF but no AgNO3 were employed. The creation of AgNPswas verified by the
color change response after a 72-hour incubation time in dark conditions. The
colorless cell filtrate solution with AgNO3 solution turned into a brown color solution.
An evaluation of the supernatant using a UV-visible spectrophotometer provided
qualitative evidence of the decrease of Ag+.Using a UV-visible spectrophotometer, the
absorbance of the sample supernatant was determined between 300 and 900 nm.
The reaction mix was then centrifuged three times for 20 minutes at 6000 rpm with
distilled water to concentrate the AgNPs. The pellet that was left behind was then
dried in a hot air furnace at 40 degree centigrade. After drying, the sample was placed
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in a glass vial together with nanoparticles that had been collected by scratching with a
sterile spatula. AgNPs were then gathered for additional characterization.
2.6. SILVER NANOPARTICLE DETECTION AND
CHARACTERIZATION
By transforming from colorless to light brown, the biosynthesized (AgNPs) in the
fungal free-cell filtrate were visually evaluated. They were further validated by a UV-
Vis spectrophotometer, scan electrons microscope (SEM), and Fourier transforms
infrared spectroscopy (FT-IR).
2.6.1. SONICATION
After it has been cleansed and centrifuged, researchers employ it in our method,
which involves using sound energy to agitate particle or discontinuity fibers that are
suspended in liquid. In most cases, frequencies higher than 20 kilohertz (kHz) are
used. Ultrasonication may be carried out with the assistance of either an ultrasonic
bath or an ultrasonic probe (sonicator). For our experiment, we used an ultrasonic
bath made by Binder in Germany (Deborah and Chung, 2017 )
2.6.2. ASSAY USING UV-VISIBLE SPECTROPHOTOMETRY
The UV-Vis spectrophotometer (CECIL (CE,7200, England )) was used to measure
the bio-decrease of Ag+ in aqueous solution after the fungal free-cell filtrate treated
with AgNO3 had been incubated for 72 hours. 0.1 ml of the filtrate was obtained
during the reduction process, diluted with deionized water, and then placed in a quartz
UV-VIS cuvette. 300 to 900 nm was the range of the scanning. Free-cell filtrate that
hadn't been altered served as a control. This was handled at the University of
Basrah's Polymer Research Center.
2.6.3. FTIR (FOURIER TRANSFORM INFRARED
SPECTROSCOPY) ANALYSIS
The free-cell filtrate underwent Fourier Transform Infrared (FTIR) (Bruker Tensor
27, Germany) examination after 72 hours of incubation. To identify the functional
groups of the stabilizing and biomolecules capping the AgNPs, FT-IR measurements
were conducted. After centrifuging the specimen solution having the nanoparticles at
5,000 rpm for 1200 second, it was filtered. The produced solid material was
subsequently crushed with potassium bromide (KBr), and pellets were created. The
pellet was examined using FTIR. This was handled at the University of Basrah's
Polymer Research Center.
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in a glass vial together with nanoparticles that had been collected by scratching with a
sterile spatula. AgNPs were then gathered for additional characterization.
2.6. SILVER NANOPARTICLE DETECTION AND
CHARACTERIZATION
By transforming from colorless to light brown, the biosynthesized (AgNPs) in the
fungal free-cell filtrate were visually evaluated. They were further validated by a UV-
Vis spectrophotometer, scan electrons microscope (SEM), and Fourier transforms
infrared spectroscopy (FT-IR).
2.6.1. SONICATION
After it has been cleansed and centrifuged, researchers employ it in our method,
which involves using sound energy to agitate particle or discontinuity fibers that are
suspended in liquid. In most cases, frequencies higher than 20 kilohertz (kHz) are
used. Ultrasonication may be carried out with the assistance of either an ultrasonic
bath or an ultrasonic probe (sonicator). For our experiment, we used an ultrasonic
bath made by Binder in Germany (Deborah and Chung, 2017 )
2.6.2. ASSAY USING UV-VISIBLE SPECTROPHOTOMETRY
The UV-Vis spectrophotometer (CECIL (CE,7200, England )) was used to measure
the bio-decrease of Ag+ in aqueous solution after the fungal free-cell filtrate treated
with AgNO3 had been incubated for 72 hours. 0.1 ml of the filtrate was obtained
during the reduction process, diluted with deionized water, and then placed in a quartz
UV-VIS cuvette. 300 to 900 nm was the range of the scanning. Free-cell filtrate that
hadn't been altered served as a control. This was handled at the University of
Basrah's Polymer Research Center.
2.6.3. FTIR (FOURIER TRANSFORM INFRARED
SPECTROSCOPY) ANALYSIS
The free-cell filtrate underwent Fourier Transform Infrared (FTIR) (Bruker Tensor
27, Germany) examination after 72 hours of incubation. To identify the functional
groups of the stabilizing and biomolecules capping the AgNPs, FT-IR measurements
were conducted. After centrifuging the specimen solution having the nanoparticles at
5,000 rpm for 1200 second, it was filtered. The produced solid material was
subsequently crushed with potassium bromide (KBr), and pellets were created. The
pellet was examined using FTIR. This was handled at the University of Basrah's
Polymer Research Center.
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2.6.4. SCAN ELECTRONS MICROSCOPE (SEM)
This electron microscopy unit at Iran's University of Tehran employed a scan
electrons microscope (SEM) (TESCAN MIRA3, French) to describe the size and
shape of AgNPs( caroling et al., 2013).
2.7. ANTIBACTERIALACTIVITY OF AGNPS
Using the agar well diffusion assay method, the potential of AgNPs was evaluated
for their antibacterial effectiveness (Perez et al., 1990). We evaluated four types of
multidrug resistant pathogenic bacteria, including Escherichia coli, Klebsiella
pneumonia, Staphylococcus aureus and Pseudomonas aeruginosa. Each overnight-
grown bacterial culture was streaked with swabs before being placed on sterile Muller-
Hinton agar (MHA) plates. Utilizing a sterilized stainless steel Cork borer, wells in agar
plates with a diameter of 5 mm were created (local,Iraq ). Two concentrations of silver
nanoparticle solutions (50 and 100 g/ml) have beenapplied to the wells. After
incubation for 1 day at 37 degree centigrade, the wells have beeninvestigated for the
existence of inhibitory zones, and the diameters of these placeshave been
determined.
2.8. ASSAY FOR DETERMINING SYNERGISTIC EFFECTS.
To assess the bactericidal effectiveness of these nanoparticles both alone and in
conjunction with antibiotics, the disk diffusion technique was utilized to measure the
synergistic effect of extracellularly generated AgNPs with routinely utilized antibiotics
(Ampicillin, Chloramphenicol) (Devi and Joshi, 2012). conventional antibiotic disks
(Ampicillin, Chloramphenicol). Standard antibiotic discs (6 mm in diameter) were
placed over the MHA medium that had been injected with test organisms after being
impregnated with 20 l of freshly made AgNPs. Positive controls were standard
antibiotic disks. Filtrate devoid of fungi served as the adverse control. For 24 to 48
hours, these plates were incubated at 37 degree centigrade. The inhibition places of
the control and treatment plates have been assessed after incubation. The assays
were all carried out in triplicate.
2.9. EVALUATION OF THE INCREASE IN FOLD AREA
The improvement in fold area was determined by comparing the mean contact area
of the inhibitory place that was formed by an antibiotic by itself and by an antibiotic in
combination with AgNPs. The fold increase area was determined utilizing the formula
(B2- A2) / A2, that A represents the inhibition place diameter generated by the activity
of antibiotics on their own, and B represents the inhibition place diameter induced by
the activity of antibiotics in combination with AgNPs (Birla et al., 2009).
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2.10.STATISTICAL ANALYSIS
We utilized the Statistical Package for the Social Sciences (SPSS), version 2020
(Copyright IBM Inc., USA). The least substantial difference (LSD) test was used in the
statistical data analysis that was performed utilizing Guide. This test was used to
compare the substantial differences that existed between the averages with a
probability threshold of p less than 0.01.
3. RESULTS
3.1. FUNGAL IDENTIFICATION
The discipline of biological science is seeing fast advancements in the use of
nanotechnology. In the course of this research, Phomatropica cell filtrate was used to
effectively produce AgNPs. The fungus was extracted from the soil and cultivated at a
temperature of 28 degrees Celsius on PDA medium. The fungus has been recognized
as Phomatium due to the features of its colony morphology (the colonies appeared
white, and with regular shape, mycelium pale white color (fig 1a), in the single colony
there was a large number of conidia deposit in the middle of the colony) (Fig. 1b) and
its micromorphology (Pycnidia, conidia subglobose, flaskshaped with conspicuous
dark circumval.
Figure 1. Morphology of Phomatropica
(a,b) Macroscopic morphology (7d and 28 degree centigrade); (c) microscopic
morphology ( 40 x).
3.2. GENETIC IDENTIFICATION OF PHOMATROPICA
3.2.1. GENOMIC DNA EXTRACTION
The technique of electrophoresis for DNA extraction under UV transilluminator
showed clear isolated DNA of Phomatropica (Figure2).
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c
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2.10.STATISTICAL ANALYSIS
We utilized the Statistical Package for the Social Sciences (SPSS), version 2020
(Copyright IBM Inc., USA). The least substantial difference (LSD) test was used in the
statistical data analysis that was performed utilizing Guide. This test was used to
compare the substantial differences that existed between the averages with a
probability threshold of p less than 0.01.
3. RESULTS
3.1. FUNGAL IDENTIFICATION
The discipline of biological science is seeing fast advancements in the use of
nanotechnology. In the course of this research, Phomatropica cell filtrate was used to
effectively produce AgNPs. The fungus was extracted from the soil and cultivated at a
temperature of 28 degrees Celsius on PDA medium. The fungus has been recognized
as Phomatium due to the features of its colony morphology (the colonies appeared
white, and with regular shape, mycelium pale white color (fig 1a), in the single colony
there was a large number of conidia deposit in the middle of the colony) (Fig. 1b) and
its micromorphology (Pycnidia, conidia subglobose, flaskshaped with conspicuous
dark circumval.
Figure 1. Morphology of Phomatropica
(a,b) Macroscopic morphology (7d and 28 degree centigrade); (c) microscopic
morphology ( 40 x).
3.2. GENETIC IDENTIFICATION OF PHOMATROPICA
3.2.1. GENOMIC DNA EXTRACTION
The technique of electrophoresis for DNA extraction under UV transilluminator
showed clear isolated DNA of Phomatropica (Figure2).
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a
b
c
Figure 2. 0.8% of agarose gel Electrophoresis showed total DNA band of Phomatropica.
3.2.2. PCR AMPLIFICATION
The results of the molecular diagnosis of the isolate after electrolysis on agarose
gel(1%) showed that the results of the DNA chain reaction using ITS1-ITS4 interfacial
primers showed the existence of a
clear bundle resulting from the process of duplication of these genes, and the
binding of the primer to its complement sequence in the DNA template ~550 bp
(Figure 3).
Figure 3. The electrophoresis of PCR.
3.2.3. SEQUENCING OF ITS GENE
The ITS nucleotidesequence is 100% homology toPhoma tropica (accession
number JF 923821.1) as registered in the GenBankdatabase. Based onmolecular and
morphological features, the fungal has been determined asPhomatropica.
3.3. SILVER NANOPARTICLE (AGNP) BIOSYNTHESIS
After 24 hours of incubation in the dark condition, P.tropica isolate's extracellular
AgNPsbiosynthesis was visually detected to modify the color of the culture
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supernatant in comparison to the control. Figures 4 and 5 displayed AgNPs harvest
derived from isolation, which ranged in hue from colorless to brown.
Figure 4. Color change observed in fungal cell filtrate (FCF) of P.tropica after exposure to
AgNO3, a- without 1mM AgNO3 b- after 24h treated with AgNO3
Figure 5. Nanoparticles synthesized byP. tropica
3.4. CHARACTERIZATION OFBIOSYNTHESISAGNPS
3.4.1. SONICATION OF AGNPS SOLUTION
Figure 6 shows how 0.001g of AgNPs were blended with 5 ml of purified water and
sonicated for 15-20 minutes using an ultrasonic bath.
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supernatant in comparison to the control. Figures 4 and 5 displayed AgNPs harvest
derived from isolation, which ranged in hue from colorless to brown.
Figure 4. Color change observed in fungal cell filtrate (FCF) of P.tropica after exposure to
AgNO3, a- without 1mM AgNO3 b- after 24h treated with AgNO3
Figure 5. Nanoparticles synthesized byP. tropica
3.4. CHARACTERIZATION OFBIOSYNTHESISAGNPS
3.4.1. SONICATION OF AGNPS SOLUTION
Figure 6 shows how 0.001g of AgNPs were blended with 5 ml of purified water and
sonicated for 15-20 minutes using an ultrasonic bath.
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Figure 6. the AgNPs solution, the right( a) one before sonication and the left (b) one after
sonication
3.4.2. UV-VISIBLE SPECTROPHOTOMETRY ANALYSIS
By employing a UV-visible spectrophotometer to conduct qualitative testing on the
supernatant, the reduction of silver ions was verified. After 24 hours, 1 ml of the
sample supernatant was removed, and the absorbance has been determined
between 300 and 900 nm utilizing a UV-visible spectrophotometer (fig.7). At 423 nm,
the absorbance peak was noted.
Figure 7. UV-Visible spectra of produced AgNPs by fungi.
3.4.3. SCAN ELECTRONS MICROSCOPE (SEM)
Images collected utilizing SEM with a magnification of 200Kx indicated that the
AgNps have been collected and generally spherical in form, with diameters ranging
from 55 to 99 nm.
0
0,6
1,2
1,8
2,4
0
225
450
675
900
Absorbance [A] - 103
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Figure 8. The biosynthesized AgNPs in the fungal free-cell filtrate were depicted in a SEM
micrograph as spherical shapes aggregated with size ranges from 55 to 99 nm (magnification
200 K X).
3.4.4. FTIR ANALYSIS
It has been proven that FT-IR experiments have the potential to detect putative
biomolecules essential in the bio removal of silver ions and the stability of AgNPs. The
FTIR spectrum study indicates that the supernatant of Phomatropica includes
biomolecules, which are responsible for the conversion of silver ions into AgNPs
(Figure 9). The results of this research also demonstrated the existence of eight
distinct stretch bands, and their values are as follows: 3358.43, 2925.48, 2845.13,
1745.26, 1645.62, 1539.88, 1455.99, and 1078.01. (cm-1).
Figure 9. FTIR spectrum of AgNPs biosynthesized by P. tropica with distinct peaks.
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Figure 8. The biosynthesized AgNPs in the fungal free-cell filtrate were depicted in a SEM
micrograph as spherical shapes aggregated with size ranges from 55 to 99 nm (magnification
200 K X).
3.4.4. FTIR ANALYSIS
It has been proven that FT-IR experiments have the potential to detect putative
biomolecules essential in the bio removal of silver ions and the stability of AgNPs. The
FTIR spectrum study indicates that the supernatant of Phomatropica includes
biomolecules, which are responsible for the conversion of silver ions into AgNPs
(Figure 9). The results of this research also demonstrated the existence of eight
distinct stretch bands, and their values are as follows: 3358.43, 2925.48, 2845.13,
1745.26, 1645.62, 1539.88, 1455.99, and 1078.01. (cm-1).
Figure 9. FTIR spectrum of AgNPs biosynthesized by P. tropica with distinct peaks.
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3.5. AGNPS HAVE ANTIBACTERIAL ACTIVITIES
The studied strains of Gram negative and Gram positive bacteria were resistant to
the biosynthesized AgNPs' antibacterial action. According to the findings, bacterial
growth was slightly less inhibited by AgNPs at a 50 g/ml concentration (0-21 mm
inhibition zones) than it was by a 100 g/ml concentration (13-25 mm inhibition zones)
(Fig.10). AgNPs had the lowest growth inhibitory activity vsK. pneumonia and the
highest vsP. aeruginosa. Without AgNPs, no inhibitory zones could be seen in the
fungal free cell filtrate (FCF). Similar findings were reported using AgNPs produced by
Papulaspora pallidula by Tawfik and Ahmad (2015).
Figure 10. The growth inhibition zones that four strains of human pathogenic bacteria
displayed in response to two nanoparticles of silver doses (AgNPs) produced by the fungus P.
tropica.
3.6. COMBINATION EFFICACY OF AGNPS WITH
AMPICILLINAND CHLORAMPHENICOL
This particular research utilizing the disk diffusion technique, we tested the
effectiveness of these AgNPs in conjunction with antibiotics at concentrations of 50
and 100 g/ml versus gram-negative and gram-positive bacteria. The width of the
inhibitory zone, measured in millimeters, surrounding antibiotic disks with and without
resistance vs test bacteria is illustrated in Fig. 11 and 12. In all of the instances, the
inhibition places diameter for antibiotics alone and in conjunction with AgNPs
demonstrated a substantial increase in fold area. This was the case with ampicillin
and chloramphenicol at concentrations of 50 and 100 µ
g/ml of AgNPs (Table 1). The
synergistic activity of AgNPs at 50 µ
g/ml concentration with antibiotics were found to
be higher vsE. Coli and P. aeruginosa as compared to S.aureusand. K. pneumonia
The synergistic activity of AgNPs at 100µg/ml concentration with antibiotics have been
detected to be greatervsP. aeruginosa and as E. coli compared to K. pneumonia
andS. aureus.
Zone of inhibi*on
0
8
15
23
30
P.tropica (Biomass)
E.Coli
K.pneumoniae
P.aeruginosa
S. aureus
13,0
21,0
0,0
13,0
15,5
25,0
13,0
13,0
100µg/ml
50µg/ml
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Table 1. Mean inhibition place (mm) brought about by various antibiotics with/withoutAgNPs
created utilizing the fungus p tropicavs the test organisms
Figure 11. Antibacterial activities of biosynthesized nanoparticles of silver at amount (50 g/ml)
vsE. coli (A), K. pneumonia (B), P. aeruginosa (C), S. aureus (D) commercial antibiotic
Ampicillin (1) and a combination of AgNPs with Ampicillin (2) commercial antibiotic
chloramphenicol (3) and a combination of AgNPs with chloramphenicol (4) Fungal cell-free
filtrate (5)
Inhibition Zone (mm )
AgNPs100µg/mlAgNPs 50µg/ml
Bacterial
strains
Incre
ase
in
fold
area
C+
AgNP
s
chlor
amph
enicol
Incre
ase
in
fold
area
Am +
AgNP
s
Ampi
cillin
Incre
ase
in
fold
area
C+
AgNP
s
chlor
amph
enicol
Incre
ase
in
fold
area
Am +
AgNP
s
Ampi
cillin
0.08325.524.5312-0.085 2524312-E. coli
0.13424.5231.0078.5-022.522.50.5637.5-
K.
pneumonia
0.92925184.06313.5-0.64120.5 162.06310.5-
P.
aeruginosa
0.32726.5230.42518.515.50.1262624.50.3491815.5S.aureus
The increase in fold area of places of inhibition has been determined by comparing the inhibition place
created by antibiotic only with the places of inhibition obtained for antibiotics paired with AgNPs manufactured
utilizing the fungal isolate.
* The values represent the averages of three replicates.
differences that are significant at P 0.01.**
-The diameter of the disc, which was measured to be 6 millimeters, was utilized in the lack of the growth of
bacteria inhibition places in order to compute the fold increase in columns 1, and 7.
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Table 1. Mean inhibition place (mm) brought about by various antibiotics with/withoutAgNPs
created utilizing the fungus p tropicavs the test organisms
Figure 11. Antibacterial activities of biosynthesized nanoparticles of silver at amount (50 g/ml)
vsE. coli (A), K. pneumonia (B), P. aeruginosa (C), S. aureus (D) commercial antibiotic
Ampicillin (1) and a combination of AgNPs with Ampicillin (2) commercial antibiotic
chloramphenicol (3) and a combination of AgNPs with chloramphenicol (4) Fungal cell-free
filtrate (5)
Inhibition Zone (mm )
AgNPs100µg/ml
AgNPs 50µg/ml
Bacterial
strains
Incre
ase
in
fold
area
C+
AgNP
s
chlor
amph
enicol
Incre
ase
in
fold
area
Am +
AgNP
s
Ampi
cillin
Incre
ase
in
fold
area
C+
AgNP
s
chlor
amph
enicol
Incre
ase
in
fold
area
Am +
AgNP
s
Ampi
cillin
0.083
25.5
24.5
3
12
-
0.085
25
24
3
12
-
E. coli
0.134
24.5
23
1.007
8.5
-
0
22.5
22.5
0.563
7.5
-
K.
pneumonia
0.929
25
18
4.063
13.5
-
0.641
20.5
16
2.063
10.5
-
P.
aeruginosa
0.327
26.5
23
0.425
18.5
15.5
0.126
26
24.5
0.349
18
15.5
S.aureus
The increase in fold area of places of inhibition has been determined by comparing the inhibition place
created by antibiotic only with the places of inhibition obtained for antibiotics paired with AgNPs manufactured
utilizing the fungal isolate.
* The values represent the averages of three replicates.
differences that are significant at P 0.01.**
-The diameter of the disc, which was measured to be 6 millimeters, was utilized in the lack of the growth of
bacteria inhibition places in order to compute the fold increase in columns 1, and 7.
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Figure 12. The inhibition places (mm) displayed by biosynthesized nanoparticles of silver at
concentrations (100g/ml) vsE. coli (A), Klebsiella pneumonia (B), P. aeruginosa (C), and S.
aureus (D) commercial antibiotic Ampicillin (1) and an AgNPs/Ampicillin combination (2)
commercial antibiotic chloramphenicol (3) and an AgNPs/Chloramphenicol combination (4)
Fungal cell-free filtrate (5).
4. DISCUSSION
4.1. ISOLATION AND IDENTIFICATION
OFPHOMATROPICAFROM SOIL
Phomatropica has been chosen for the production of nanoparticles because it is
simple to extract from soil, it is straightforward to cultivate on straightforward media
such as PDA, and most importantly, it has consistent biochemical properties ( Rai et
al, 2009 ). In addition, there is no research done so far on selecting Phomatropica for
the production of AgNPs. This is something that has to be done.
(Boerema et al., 2004) conducted research on the Phoma species based on the
physical and cultural aspects of each species. The recognition of Phoma depending
only on morphological characteristics was relatively inconsistent, which contributed to
confusion over its identity. Because of this, molecular-based approaches were used,
which turned out to be a superior choice for the detection and investigation of genetic
differences amongst fungi (deGruyter et al., 2009; Aveskamp et al., 2010).
Several of the most frequent options for phylogenetic inference at the genus level
or lower is bi-parental, nuclear ITS regions. This is because these areas have a
greater rate of base replacement than the genes found in most organelles. The
phylogenetic connections between Phoma and the groups to which it is closely related
were examined in great detail utilizing ITS sequence data ( Iryini et
al,2009 :Aveskamp et al, 2009).
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4.2. BIOSYNTHESIS AND CHARACTERIZATIONOF AGNPS
Since dangerous bacteria have been showing signs of antibiotic resistance during
the past ten years, researchers are concentrating on creating new antibacterial
substances. Ag-NPs as antibacterial agents have emerged as viable candidates in the
current medical landscape (Duran et al. 2007). AgNPs can be produced by
microorganisms like fungus, which has significant promise for numerous applications
(Alghuthaymiet al., 2015). Due to their capacity to create AgNPs, many Phoma
species were shown to be able to synthesis nanoparticles of silver (Aniketet al, 2013 ;
Sudhiret al,2016 ; Aniketet al, 2011).This is the first account of the environmentally
friendly extracellular production of AgNPs by P. tropica. As evidenced by the color shift
from colorless to brownish after 3 days of incubation after being exposed to a solution
of 1 mM AgNO3, the current investigation demonstrated that the chosen fungus, P.
tropica, displayed a great potential for the AgNPs synthesis in culture medium. These
results are consistent with earlier research utilizing several fungi species (Asemet al.
2017;Tejal Barkhade.. 2018 ;Bahimbaet al., 2011: Mohamed et al, 2021).
The addition of AgNO3 to the fungal free-cell filtrate caused a color shift as a result
of the excitation of silver's surface plasmon resonance vibration that verified the
decrease of silver ions as reported by (Chitra and Annadurai,2013).
4.3. UV-VISIBLE SPECTROPHOTOMETRY ANALYSIS
The current research demonstrated that UV-Vis spectrophotometry examination
revealed a maximum with great absorbance at 423 nm, which stated that the
investigated fungus had been replicating AgNPs, indicating that the production of
AgNPs has been complete after 3 days of incubation with free-cell filtrate. This is in
line with a few other pieces of art that have been done (Mohamed et al 2021; Aniket et
al, 2013 ).
When compared to previous studies, it appears that there have been some
variances in the features of the AgNPs generated by distinct species of fungus. These
variations have been seen in the AgNPs (Birla et al. 2009; Chitra and Annadurai,
2013; Maliszewska et al., 2009; Raheman et al., 2011). These variations might be
attributable to the origin of the fungal isolates or strains as well as the environment
under which they grew (Marambio-Jones and Hoek, 2012). Based on the findings of
Neethu et al. (2018), the amount of fungal mycelium was directly correlated to the
amount of AgNP that was synthesized.
4.4.BIOSYNTHESIZED DESCRIPTION AGNPS BY SCAN
ELECTRONS MICROSCOPY (SEM)
Different reaction parameters affect the form and size of biosynthesized
nanoparticles of silver in solution.
The AgNPsbiosynthesized morphology by the chosen fungus was scattered with
size of 55-90 nm, as shown by SEM ostly spherical and pictures.
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4.2. BIOSYNTHESIS AND CHARACTERIZATIONOF AGNPS
Since dangerous bacteria have been showing signs of antibiotic resistance during
the past ten years, researchers are concentrating on creating new antibacterial
substances. Ag-NPs as antibacterial agents have emerged as viable candidates in the
current medical landscape (Duran et al. 2007). AgNPs can be produced by
microorganisms like fungus, which has significant promise for numerous applications
(Alghuthaymiet al., 2015). Due to their capacity to create AgNPs, many Phoma
species were shown to be able to synthesis nanoparticles of silver (Aniketet al, 2013 ;
Sudhiret al,2016 ; Aniketet al, 2011).This is the first account of the environmentally
friendly extracellular production of AgNPs by P. tropica. As evidenced by the color shift
from colorless to brownish after 3 days of incubation after being exposed to a solution
of 1 mM AgNO3, the current investigation demonstrated that the chosen fungus, P.
tropica, displayed a great potential for the AgNPs synthesis in culture medium. These
results are consistent with earlier research utilizing several fungi species (Asemet al.
2017;Tejal Barkhade.. 2018 ;Bahimbaet al., 2011: Mohamed et al, 2021).
The addition of AgNO3 to the fungal free-cell filtrate caused a color shift as a result
of the excitation of silver's surface plasmon resonance vibration that verified the
decrease of silver ions as reported by (Chitra and Annadurai,2013).
4.3. UV-VISIBLE SPECTROPHOTOMETRY ANALYSIS
The current research demonstrated that UV-Vis spectrophotometry examination
revealed a maximum with great absorbance at 423 nm, which stated that the
investigated fungus had been replicating AgNPs, indicating that the production of
AgNPs has been complete after 3 days of incubation with free-cell filtrate. This is in
line with a few other pieces of art that have been done (Mohamed et al 2021; Aniket et
al, 2013 ).
When compared to previous studies, it appears that there have been some
variances in the features of the AgNPs generated by distinct species of fungus. These
variations have been seen in the AgNPs (Birla et al. 2009; Chitra and Annadurai,
2013; Maliszewska et al., 2009; Raheman et al., 2011). These variations might be
attributable to the origin of the fungal isolates or strains as well as the environment
under which they grew (Marambio-Jones and Hoek, 2012). Based on the findings of
Neethu et al. (2018), the amount of fungal mycelium was directly correlated to the
amount of AgNP that was synthesized.
4.4.BIOSYNTHESIZED DESCRIPTION AGNPS BY SCAN
ELECTRONS MICROSCOPY (SEM)
Different reaction parameters affect the form and size of biosynthesized
nanoparticles of silver in solution.
The AgNPsbiosynthesized morphology by the chosen fungus was scattered with
size of 55-90 nm, as shown by SEM ostly spherical and pictures.
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Numerous studies have noted that different fungus species, pH levels, and
temperatures affect the AgNPs shape and size that are generated (Martinez-
Castanonet al., 2008; Marambio-Jones and Hoek, 2010; Muhsin and Hachim,
2015 ;Aniketet al, 2013 ).
4.5. FT-IR ANALYSISA OF THE BIOSYNTHESIZED AGNPS
P. tropica has been shown to contain biomolecules that turn silver ions into AgNPs.
These biomolecules occur in 8 different stretching bands, which are: 3358.43,
2925.48, 2845.13, 1745.26, 1645.62, 1539.88, 1455.99, 1078.01, and others (cm-1).
The distinctive hydrogen-linked OH set that could be the result of the formation of
nanoparticles in an aqueous phase, is connected to the bending vibrations of the OH
alcohol bonds, phenols, and the N-H stretching vibration of main protein amides, all of
which make a contribution to the peak at 3358.43cm-1. These vibrations are
responsible for the existence of the peak.
The C-H stretching associated with the methylene protein set and the N-H
stretching associated with the amine salt are both candidates for the causes of the
maxima at 2925.48 and 2845.13 cm-1, respectively. This finding is undeniably linked
to the modification of the electric surroundings of the methylene and methane sets
that was brought about by the close proximity of the AgNPs and carbonyl. The
stretching vibrations of C=O was the source of the experimental group that could be
seen at 1745.26 cm-1. In the FTIR spectrum, there are two bands that are visible.
These bands have been connected with the stretching vibration of the amide I band
and the amide II band of the protein, respectively. The bands have a wavelength of
1645.62 cm-1 and 1539.88 cm-1. (Joshi,2012) suggests that the absorption peak at
1455.99 cm-1 might be related to geometric bending vibration of amino acid residue
sets with free protein carboxylate sets -COO- (carboxylate ion), 1078.01 (ethers,
esters, and C-O alcohol stretched oxalic acids), and C-N stretched of aliphatic
amines.
4.6. AGNPSANTIBACTERIAL ACTIVITY ON PATHOGENIC
BACTERIA
At two different doses (100 g/ml and 50 g/ml), the biosynthesized AgNPs were
shown to limit the development of human pathogenic bacterial strains, which were
examined. This was another finding from the present investigation. In contrast, the
effectiveness of AgNPs as an antibacterial agent was inconsistent. Even though the
mechanism by which AgNPs prevent bacteria from growing is not extensively
established, it is possible that it is linked to the impact of Ag+ by leading to damage of
bacterial cell membranes, the damage of enzymes, or the conformational changes of
DNA. This is what has been proposed by other study results (Kim et al. 2007;
Marambio-Jones and Hoek, 2010).
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4.7. COMBINATION EFFICACY OF AGNPS WITH AMPICILLIN
AND CHLORAMPHENICOL
According to increased fold area, the biosynthesized AgNPs and the medicines
ampicillin and chloramphenicol significantly boosted efficiency vs the chosen human
pathogenic bacteria ( Birla et al. 2009). These results are consistent with those of
earlier research that investigated the synergic impact of AgNPs produced from a
variety of fungal species when used in conjunction with a selection of various
commercialized antibiotics and put to the test versus Gram-negative and Gram-
positive bacteria (Fayaz et al., 2010; Devi and Josh, 2011; Gudikandula et al., 2015;
Shareef et al., 2017).
(Fayaz et al. 2010) also showed an increase in the antibacterial activity of
chloramphenicol, erythromycin, kanamycin, and ampicillin when used in conjunction
with AgNPsvs Salmonella typhi, Escherichia coli, Staphylococcus aureus, and
Micrococcus luteus.
(Devi and Joshi, 2011) showed an improvement in the antibacterial activity of
ciprofloxacin, chloramphenicol, erythromycin, and methicillin when combined with
biosynthesized AgNPs versus Enterococcus faecalis, Salmonella enterica,
Streptococcus pyogenes, and Staphylococcus aureus.
5. CONCLUSION
The production of AgNPs using P. tropicawas investigated in the current study this
finding is the first for this fungus species in Iraq. The generated AgNPs demonstrated
activity versus Gram-positive and Gram-negative human pathogenic bacteria
throughout a wide range. This fungus shows potential as a natural source for the
synthesis of AgNPs, which have applied in the medical product and pharmaceutical
manufacturing industries.
6. ACKNOWLEDGMENT
We appreciate the Basrah University (Iraq) administration's support of this research
project as a requirement for the MSc.
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(3) Aniket. G*, Swapnil. G*, Nelson.D†andMahendra.R. (2013). Screening of
different species of Phomafor Synthesis of Silver nanoparticles. 13, 1-33.
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314
4.7. COMBINATION EFFICACY OF AGNPS WITH AMPICILLIN
AND CHLORAMPHENICOL
According to increased fold area, the biosynthesized AgNPs and the medicines
ampicillin and chloramphenicol significantly boosted efficiency vs the chosen human
pathogenic bacteria ( Birla et al. 2009). These results are consistent with those of
earlier research that investigated the synergic impact of AgNPs produced from a
variety of fungal species when used in conjunction with a selection of various
commercialized antibiotics and put to the test versus Gram-negative and Gram-
positive bacteria (Fayaz et al., 2010; Devi and Josh, 2011; Gudikandula et al., 2015;
Shareef et al., 2017).
(Fayaz et al. 2010) also showed an increase in the antibacterial activity of
chloramphenicol, erythromycin, kanamycin, and ampicillin when used in conjunction
with AgNPsvs Salmonella typhi, Escherichia coli, Stap