Novel Research in Microbiology Journal (2021), 5(3): 1256-1268 (Print) (ISSN 2537-0286)

Research Article (Online) (ISSN 2537-0294)

www.nrmj.journals.ekb.eg
DOI: 10.21608/nrmj.2021.178303

Extracellular biosynthesis of silver nanoparticles using Bacillus subtilis and their

antibacterial activity against clinical bacterial species

Nouran H. Assar1*; Aya allah T. Mohamed2,3; Rehab M. Abd El-Baky2,3; Reham Ali Ibrahem2
1
Department of Microbiology, General Division of Basic Medical Sciences, Egyptian Drug Authority former
National Organization for Drug Control and Research (NODCAR), Giza, Egypt; 2Department of Microbiology
and Immunology, Faculty of Pharmacy, Minia University, Minia, Egypt; 3Microbiology and Immunology
Department, Faculty of Pharmacy, Deraya University, Minia, Egypt

*
Corresponding author E-mail: drnouranhamed@hotmail.com

Received: 17 May, 2021; Accepted: 15 June, 2021; Published online: 17 June, 2021

Abstract

The aims of this study were to biosynthesize silver nanoparticles (AgNPs) using Bacillus
subtilis supernatant, and to evaluate their in vitro antibacterial potential against human
pathogens; namely Staphylococcus aureus (Staph. aureus) and Escherichia coli (E. coli).
Nanoparticles (NPs) are becoming popular in different fields of research, and are useful in
combating vast number of microbial diseases. NPs may be artificially synthesized in vitro
using chemical methods and\or via extracellular metabolites produced by the bacterial strains.
Copyright policy
In the present study, biosynthesis of AgNPs was carried out in vitro using supernatants of B.
NRMJ allows the subtilis. Biosynthesized AgNPs were characterized through several physical methods. The
author(s) to hold the recorded Z-average (d. nm) was 135.0 nm; with 99.2 % of the NPs displaying a hydrodynamic
copyright, and to
retain publishing distance across of 188.0 nm (SD= 117.7). The polydispersity index was 0.246 and the Zeta-
rights without any potential value was - 17.2 mV, which indicates good colloidal stability. Results of the
restrictions. This work
Transmission electron microscope (TEM) observation indicated that the particles were
is licensed under
https://creativecommo spherical in shape with an average size of 21.8- 27.5 nm. The antibacterial efficacy of the
ns.org/licenses/by/4.0/ AgNPs against Methicillin resistant Staph. aureus (MRSA) and E. coli clinical isolates was
evaluated in vitro using the agar well diffusion. The AgNPs demonstrated antibacterial
potential against MRSA and E. coli isolates; recording 18 and 15 mm diameter of zones of
inhibition, respectively. The minimum inhibitory concentration (MIC) was found to be 142
µg/ ml, while the recorded minimum bactericidal concentration (MBC) was 284 µg/ ml. The
mode of action of the AgNPs was investigated using the Scanning electron microscope (SEM),
which was recognized as bacterial cell lysis and elongation. Current data suggest an efficient
biosynthesis of stable AgNPs by B. subtilis with remarkable antibacterial potential.

Keywords: Bacillus subtilis, Biosynthesis, Silver nanoparticles, MRSA, E. coli

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1. Introduction

Methicillin-resistant Staph. aureus (MRSA) is During the current work, the ability of B. subtilis
known as a serious causal agent of nosocomial strain to cause extracellular synthesis of AgNPs using
infections that spreads worldwide, and has a negative 1 mM AgNO3 as a precursor solution was
impact on the patient's health resulting in huge demonstrated. These biosynthesized AgNPs exhibited
increases in the health care costs (Luteijn et al., 2011). the power to counteract the clinical strains of E. coli
Staph. aureus acquires methicillin resistance by and Staph. aureus. The objectives of this study were to
insertion of staphylococcal cassette chromosome investigate the ability of B. subtilis to synthesis
(SCCmec) carrying the mecA gene into the microbial AgNPs, and to test the antibacterial efficacy of the
genome. This gene encodes for penicillin-binding biosynthesized AgNPs against E. coli and MRSA.
protein PBP-2a, which is not inhibited by the pre-
existing β-lactams antibiotics (Makgotlho et al., 2009). 2. Materials and methods

In recent years, nanotechnology has been of great 2.1. Clinical standard strains and bacterial isolates
interest due to its high impact in several fields such as;
2.1.1. Standard bacterial strains
energy, medicine, electronics and space industries.
Medical applications of biosynthesized AgNPs have In this study, B. subtilis ATCC (6633), E. coli
already started including; targeted drug delivery, ATCC (8739) and Staph. aureus ATCC (6538) were
cancer treatment, gene therapy and DNA analysis, kindly provided by Microbiology department,
antibacterial agents, biosensors and enhancers of Egyptian Drug Authority (EDA), Giza, Egypt.
reaction rates.
2.1.2. Collection and identification of the clinical
Many microbes such as bacteria, fungi, bacterial isolates
actinobacteria and yeasts are capable of intra-cellular
or extra-cellular biosynthesis of NPs, mineral crystals During a 12-month period (July, 2017 - l July,
and metallic NPs (Li et al., 2011). In addition, the 2018), clinical bacteria were isolated from different
most widely used type of nanoparticles is the AgNPs; patient's wounds and urine samples; recovered from a
manipulated as novel therapeutic agents due to their total of 96 male and female patients of varying ages,
antibacterial; antifungal, antiviral and anti- administered to the Microbiology Laboratories of El
inflammatory potencies. Kasr El Aini hospital, Cairo, Egypt. Identification of
these bacterial isolates was carried out using Gram
Metallic nanoparticles have become of high staining and several standard biochemical reactions
importance due to their application in many fields, including; catalase assay, coagulase assay, DNase test,
especially when being synthesized in an Mannitol utilization, Lactose fermentation and growth
environmentally safe and inexpensive way. There are on a number of specific media such as; Eosin
numerous microorganisms possessing the ability to methylene blue agar, Triple sugar ion agar and
synthesize NPs (Pantidos and Horsfall, 2014), as they Simmon citrate agar (Bergey and Holt, 2000). After
have the ability to reduce the Ag+ to AgNPs (Fayaz et that, Vitek 2 identification system was manipulated
al., 2010). The previous work of Panacek et al., (2006) using ID-Gram Positive Cocci cards (ID-GP cards;
revealed that AgNPs have effective antimicrobial bioMe´rieux) and ID -Gram Negative cards (ID-GN
activity against both of Gram-positive and Gram- cards; bioMe´rieux), according to the manufacturer’s
negative bacteria; including the highly multi-resistant instructions (Wattal and Oberoi, 2016).
strains such as MRSA.
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2.2. Antibiotic susceptibility assay In this study, B. subtilis ATCC (6633) was grown
in 250-ml Erlenmeyer flask containing 100 ml Luria-
The recovered Staph. aureus and E. coli isolates Bertani broth (LB) medium for 36 h at 37 ºC with
were tested for their antibiotic sensitivity using two shaking at 150 rpm. After incubation, the culture was
antibiotic disks mainly: ceftazidime (CAZ: 30 μg); centrifuged at 5,000 rpm for 30 min., and then the
cefoxitin (FOX: 30 μg); in addition to amikacin (AK: supernatant was collected to be used as the starting
30 μg); streptomycin (S: 10 μg); tetracycline (TE: 30 material for extracellular synthesis of AgNPs.
μg); amoxicillin/clavulanic acid (AMC: 30 μg); Afterwards, 1 mM of AgNO3 (1% v/v) was added to
meropenem (MEM: 10 μg); ofloxacin (OFX: 5 μg) B. subtilis supernatant (pH adjusted to 8.5), and then
and trimethoprime-sulfamethoxazole (STX: 25 μg) for incubated with shaking at 40 °C (200 rpm) for 5 d
phenotypic detection of MRSA, according to Kirby- under dark conditions (Sunkar and Nachiyar, 2012).
Bauer agar disc diffusion assay of Bauer et al., (1966). An un-inoculated broth medium was used as a control,
Results were evaluated following the criteria of the to check for the role of bacteria in the biosynthesis of
Clinical and Laboratory Standards Institute (CLSI). NPs.
(2017).
2.5. Characterization of the biosynthesized AgNPs
2.3. Detection of mecA gene for MRSA by
Polymerase chain reaction (PCR) 2.5.1. Particle sizing measurements

The presence of mecA gene was detected in Staph. Particle size analysis was conducted by means of
aureus isolates that were recorded to be resistant to dynamic light scattering assay (Laser diffractometry)
cefoxitin, through amplifying a 293-bp fragment using using an extremely compact optical bench; the CILAS
primer pair mecA forward (5’- 1064 integrates 2 sequenced laser sources pointed at 0º
ACGAGTAGATGCTCAATATAA-3’) and mecA and 45º. Measurements were taken in the range of
reverse (5′- CTTAGTTCTTTAGCGATTGC3’), in 0.04-500 μm.
reference to Sabet et al., (2007). The chromosomal
DNA was prepared from overnight cultures of the 2.5.2. Transmission electron microscope (TEM)
bacterium grown on Brain Heart Infusion (BHI) broth. The TEM was used for detection of the NPs size
DNA amplification was carried out in a PCR and shape. Samples were prepared by placing a drop
thermocycler with the following thermal cycling of the biosynthesized AgNPs over a gold-coated
profile: initial denaturation at 94ºC for 3 min. followed negative grid followed by evaporation of the solvent
by 30 sec. at 94 ºC, 30 sec. at 60 ºC and 30 sec. at 72
(Germain et al., 2003); using TEM, JEOL-JEM-1011
ºC. Denaturation, annealing and extension were
instrument at 100 kV.
performed in 30 cycle followed by an extra-cycle
of annealing at 60 °C for 30 sec., and a final extension 2.5.3. Zeta potential measurement (ZP)
at 72 ºC for 5 min. A volume of 2 µl of prepared DNA
was added to a final volume of 25 µl PCR mixture Measurement of ZP was carried out for the AgNPs
containing: 12.5 µl Master Mix (Biomatik, Canada), 1 to detect their colloidal stability, using Malvern
µl of each primer and 8.5 μl of sterile dist. water. The Zetasizer Nano ZS analyzer at room temperature
amplified products were visualized by electrophoresis, (Zetasizer Nano-ZS-instrument, Worcestershire,
with UV in 2 % agarose gels stained with Ethidium United Kingdom), in reference to Elbeshehy et al.,
bromide. (2015).

2.4. Extracellular biosynthesis of AgNPs 2.6. Detection of the in vitro antibacterial potential
of the AgNPs

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The agar well diffusion assay was used to evaluate Morphological changes in shapes of the bacterial
the in vitro antibacterial efficacy of the biosynthesized strains i.e. Staph. aureus and E. coli treated with the
AgNPs, according to Singh et al., (2013). A single biosynthesized AgNPs were observed using Scanning
colony of the tested B. subtilis was picked up, electron microscopy (SEM) (JEOL, JSM-5200 LV
inoculated into MH broth and then incubated overnight SEM, Japan); with the voltage set to 25 kV at 5,000×
at 37 °C. The OD of the bacterial suspension was magnification power, according to Chakravarty and
adjusted to 0.5 McFarland (≈1 × 108 cfu/ ml); a loopful Banerjee, (2008). The isolates were incubated
of this suspension was inoculated into Mueller Hinton individually overnight with sub-inhibitory
agar (MHA) medium, and then poured into Petri concentration of the AgNPs. At the end of incubation,
plates. A 50 µl of 1mM of AgNPs solution was added morphological changes in the bacterial cells treated
to the center of each well (10 mm), which were with AgNPs were observed under SEM, compared to
punched in the MHA plates using a sterile cork borer. the untreated control cells.
Control sample was made using either 50 µl of the
sterile supernatant derived from B. subtilis culture or 2.9. Statistical analysis
1mM of AgNO3 solution; instead of the tested AgNPs.
The significance of the antibacterial potency of
After incubation at 37 °C for 24 h, the zones of the AgNPs was checked by standard variance of
inhibition were measured using a calibrated ruler. The analysis (ANOVA). p values< 0.05 were considered
assay was performed in triplicates, and repeated two significant. The mean values ± SD (standard
times. deviation) were presented. The data was analyzed with
2.7. Evaluation of the minimum inhibitory Graph Pad Prism 7.
concentration (MIC) and minimum bactericidal
3. Results
concentration (MBC) of the AgNPs
3.1. Isolation and identification of the clinical
The MIC of the AgNPs was determined through
bacterial isolates
the two-fold broth dilution method using the MHB
following the criteria of CLSI. (2017). From a stock In this study, about 50 isolates of Staph. aureus
suspension of 1mM of the biosynthesized AgNPs; and 46 isolates of E. coli were recovered from patients
two-fold serial dilutions were prepared using MHB. of El Kasr El Aini Hospital, Microbiology
Bacterial isolates, in their exponential growth phase; laboratories, Egypt. Species-level identification of the
were inoculated in the wells of a 96-microwell plate isolates was confirmed using the biochemical profile
containing either MHB alone as a positive control, or and VITEK 2 system; with confidence levels of 93-95
MHB containing the biosynthesized AgNPs at a final % probability.
concentration ranging from 17.75- 568 µg/ ml. The
microplate was then incubated at 37 °C for 24 h. The 3.2. Antibiotic susceptibility pattern
MIC corresponded to the AgNPs concentration that
Results showed that Staph. aureus isolates were
inhibited the bacterial growth, compared to the
resistant to amoxicillin/clavulanic acid by 100 %, and
positive control. On the other hand, MBC was
presented much lower resistance towards
determined by sub-culturing the contents of the clear
trimethoprime-sulfamethoxazole by 34 %, as
wells that showed no growth in the MIC assay onto
demonstrated in Fig. (1). As demonstrated in Fig. (2),
MHA plates. The assays were performed in triplicates,
E. coli isolates were found to be resistant to
and repeated two times.
ceftazidime by 94 %, with much lower resistance to
2.8. Morphological changes in the bacterial cells Amikacin by 24 %.

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120
Susceptibility percentage (%)

100

80

60 Sensitive

40 Intermediate
Resistant
20

0
TE CAZ OFX AK STX AMC FOX
Antibiotics

Fig. 1: Antibiotic susceptibility pattern of Staph. aureus, demonstrating different sensitivity to the various tested antibiotics

120
Susceptibility percentage (%)

100

80

60 Sensitive
Intermediate
40
Resistant
20

0
S TE CAZ OFX AK STX MEM AMC
Antibiotics

Fig. 2: Antibiotic susceptibility pattern of E. coli, demonstrating different sensitivity to the various tested antibiotics

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3.3. Detection of mecA gene in the MRSA isolate of the mecA gene in these isolates using PCR assay.
Results showed that 62 % (31/50) of Staph. aureus
Further identification of the Staph. aureus isolates isolates possessed mecA gene Fig. (3).
that expressed resistance to cefoxitin, and
phenotypically identified as MRSA, through detection

Fig. 3: Agarose gel electrophoresis of PCR product obtained from the MRSA isolates. Where; Lane M: marker 100 bp ladder,
Lane 1, 2, 3, 4, 5 and 7: positive mecA gene, Lane 6: negative mecA gene

3.4. Biosynthesis of AgNPs using supernatant of B. solution from pale yellow to reddish brown (Fig. 4a,
subtilis b). This finding indicated the formation and deposition
of AgNPs, while the color of AgNO3 solution
Supernatant collected from B. subtilis LB culture used (negative control) remained unchanged.
as the starting material for extracellular synthesis of
AgNPs, turned the color of the treated AgNO3

a b

Fig. 4: (a) Supernatant culture of B. subtilis before incubation with the AgNO3 solution (yellow), and (b) supernatant culture of
B. subtilis after incubation with AgNO3 (brown), indicating the formation of AgNPs

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3.5. Physical characterization of AgNPs Zetasizer analyzer, respectively. Results in Fig. (6)
demonstrates that the Z-Average (d. nm) was 135.0
Samples of AgNPs were subjected to TEM nm; with 99.2 % of the particles exhibiting a
analysis for characterization. Results revealed that the hydrodynamic diameter of 188.0 nm (SD= 117.7). The
AgNPs were spherical in shape with particle size polydispersity index was 0.246 and the zeta-potential
diameter of 21.8- 27.5 nm, as demonstrated in Fig. (5). (ZP) value was -17.2 mV, which indicates good
The size distribution and zeta potential of AgNPs were colloidal stability of the AgNPs.
determined by dynamic light scattering and

Fig. 5: TEM micrograph at 20,000 times magnification recorded from a drop-coated film of an aqueous solution of AgNPs,
showing the spherical shape of the particles and their average diameter of 21.8- 27.5 nm

Fig. 6: Size distribution intensity graph of biosynthesized AgNPs, demonstrating Z-Average (d. nm) of 135.0

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3.6. Antibacterial efficacy of the AgNPs Fig. (7) show the inhibitory activity of AgNPs against
MRSA and E. coli isolates; recording diameter of
Preliminary screening of the in vitro antibacterial zones of inhibition of 18, 15 mm, respectively. These
potential of the AgNPs was assessed against 77 clearly indicate that the difference in the inhibitory
clinical isolates (31 MRSA and 46 E. coli), in addition action of the AgNPs on the tested bacterial spp. was
to the control standard strains of E. coli ATCC (8739) statistically significant (p< 0.05), compared to the
and Staph. aureus ATCC (6538); using the agar well control wells that showed no inhibition zones.
diffusion. The obtained data presented in Table (1) and

Table 1: Antibacterial potency of biosynthesized AgNPs, 1mM AgNO 3 and B. subtilis culture supernatant against
clinical and standard bacterial isolates

Mean zone of inhibition ± SD (in mm)

Clinical / Standard Biosynthesized AgNPs AgNO3 B. subtilis
isolates (1 mM) (1 mM) culture supernatant
MRSA 18± 1.387 10.7± 0.763 0
E. coli 15± 1.201 10.5± 0.863 0
E. coli 17± 0.112 11± 0.101 0
ATCC (8739)
Staph. aureus 19± 0.121 10± 0.112 0
ATCC (6538)
Where; Data were expressed through measurement of diameter of inhibition zones as means of three replicates in mm ± SD,
*p-value was significant < 0.05

20
Mean of zones of inhibition (mm)

18
16
14
12
10 Biosynthesized AgNPs (1mM)
8
AgNO3 (1mM)
6
4 B. subtilis culture supernatant
2
0
Clinical MRSA Clinical E. coli E. coli ATCC Staph. aureus
(8739) ATCC (6538)
Clinical\ Standard isolates

Fig. 7: Antibacterial efficacy of biosynthesized AgNPs (1 mM), AgNO3 (1 mM ) and B. subtilis culture supernatant against the
clinical and standard bacterial isolates

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3.7. The MIC and MBC of the biosynthesized 3.8. Morphological changes in shapes of the AgNPs
AgNPs treated bacterial cells

The MIC and MBC values of the biosynthesized Results of SEM indicated that the non-treated
AgNPs against the tested microbial isolates were bacterial cells were intact without any noticeable
determined. The recorded MIC was 142 µg/ ml, while damage; while upon interaction with the AgNPs, the
the MBC was 284 µg/ ml, as presented in Table (2). bacterial cells were damaged that was observed as cell
The MIC and MBC indicated massive antibacterial lysis of Staph. aureus and cell elongation of E. coli.
effectiveness of the AgNPs. (Fig. 8).

Table 2: The MIC and MBC of the biosynthesized AgNPs against the clinical and the standard bacterial isolates

Clinical / Standard isolates MIC (µg/ ml) MBC (µg/ ml)

MRSA 142 284
E. coli 142 284
E. coli ATCC (8739) 35.5 70
Staph. aureus ATCC (6538) 35.5 70

a b

c d
Fig. 8: Scanning electron micrographs of (a): control Staph. aureus cells, (b): AgNPs treated Staph. aureus cells (c): control E.
coli cells and (d): AgNPs treated E. coli cells, demonstrating cell lysis and cell elongation of Staph. aureus and E. coli,
respectively

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4. Discussion stated that low particles size is useful for successful

delivery of the targeted drugs, which comply with the
Currently, MRSA is a major cause of morbidity current detected particle size of the biosynthesized
and mortality worldwide (de Kraker et al., 2011). AgNPs that ranged from of 21.8- 27.5 nm. In a
There are several antimicrobial susceptibility testing previous study conducted by Cho et al., 2005), (it was
assays used for detection of MRSA including; proved that high surface area to volume ratio of the
oxacillin screening test, oxacillin and/or cefoxitin disk AgNPs causes high bactericidal efficacy compared to
diffusion method and oxacillin MIC test, as the bulk silver metal. Accordingly, the biosynthesized
highlighted by Tubbicke et al., (2012). Current results AgNPs are considered as promising therapeutic agents
revealed that the tested Staph. aureus isolates were that demonstrate significant antimicrobial activities.
100 % resistant to cefoxitin, so these isolates were
phenotypically identified as MRSA. These An early study conducted by Saifuddin et al.,
conventional antimicrobial tests used for MRSA (2009) revealed that AgNPs biosynthesized by reaction
identification were always associated with false of Ag+ ions with B. subtilis culture supernatant is
negative and positive results. Therefore, it is necessary exceptionally stable, which is likely attributed to
to use PCR for microbial identification, which is capping with proteins secreted by the bacteria. In
considered as a DNA-based assay. Our results addition, Zeta potential of the biosynthesized AgNPs
demonstrated that only 62 % (31/50) of Staph. aureus indicated their stability; which is also attributed to the
isolates possessed mecA gene; however, there is no presence of microbial proteins that cover the NPs. This
mecA gene detected in methicillin-sensitive Staph. coincides with the current results recording a zeta-
aureus (MSSA) strains. A previous study conducted potential value of -17.2 mV.
by Hallin et al., (2003) reported that detection of this
The antibacterial efficacy of AgNPs is attributed
gene in any strains of Staph. aureus is indicative of
to different possible mechanisms of action. Marambio-
MRSA.
Jones and Hoek, (2010) believed that the positive
Synthesis of AgNPs is accompanied with charged silver ions (Ag+) of AgNPs becomes attached
significant modifications of the properties of metals; to the negatively charged cell surface causing
owing to their extremely small size and high surface disturbance in its physical and chemical properties,
area to volume ratio. Moreover, Mohd Yusof et al., which in turn disturb the membrane functions
(2019) added that NPs exhibit distinctive including; permeability, electron transport and
characteristics that led to significant differences in respiration. Rajeshkumar and Malarkodi, (2014)
their properties compared to the bulk counterparts. proposed another possible mode of action stating that
AgNPs can interact with the microbial cells leading to
Usually, AgNPs synthesis is detected through a inhibition of their respiratory chain enzymes and
color change of the reaction mixture from yellow to increasing the cells permeability to phosphate and
dark brown (Karbasian et al., 2008), so appearance of protons; through altering trans-membrane electron
brown color indicates the formation of AgNPs, which transfer. Furthermore, AgNPs may cause damage to
is in accordance with the results of this study. A the bacterial cells in consequence of the interaction
previous work of Yamal et al., (2013) attributed this with the proteins, DNA and other sulfur- and
color change to the surface plasma resonance property phosphorus-containing cell constituents, as reported by
of the NPs. The antibacterial potency of AgNPs Nayak et al., (2015). This is the nearest mode of action
increased considerably with the decrease in the of AgNPs that our current results comply with, where
particles size. Dauthal and Mukhopadhyay (2016)

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cell lysis of Staph. aureus and cell elongation in E. coli Bergey, D.H. and Holt, J.G. (2000). Bergey's Manual
were observed upon treatment with AgNPs. of Determinative Bacteriology, Philadelphia
Lippincott Williams and Wilkins.
Conclusion
Chakravarty, R. and Banerjee, P.C. (2008).
In this study, the ability for extracellular Morphological changes in an acidophilic bacterium
biosynthesis of AgNPs using 1 mM AgNO3 as a induced by heavy metals. Extremophiles. 12(2): 279-
precursor solution was demonstrated by the B. subtilis. 284.
The AgNPs had the power to counteract with clinical
isolates of E. coli and Staph. aureus. TEM analysis Cho, K.H.; Park, J.E.; Osaka, T. and Park, S.G.
revealed the spherical shape of AgNPs with particle (2005). The study of antimicrobial activity and
size of 20- 47 nm. Zeta potential analysis presented a preservative effects of nanosilver ingredient.
value of -17.2 mV, which expresses the colloidal Electrochimica Acta. 51(5): 956-960.
stability of the AgNPs. Such NPs have been found to
Clinical and Laboratory Standards Institute
be effective against different drug-resistant bacterial
(CLSI). (2017). Performance Standards for
strains that were pathogenic, and these findings were
Antimicrobial Susceptibility Testing, in 27th Edition.
confirmed through SEM analysis. The possible
CLSI supplement M100, Wayne, PA: Clinical and
antibacterial activity of AgNPs against the different
Laboratory Standards Institute.
pathogens was confirmed during this study.
Dauthal, P. and Mukhopadhyay, M. (2016). Noble
Acknowledgements
metal nanoparticles: plant-mediated synthesis,
The authors appreciate the support provided by the mechanistic aspects of synthesis, and applications.
Egyptian Drug Authority (EDA), Formerly National Industrial and Engineering Chemistry Research. 55:
Organization for Drug Control and Research 9557-9577.
(NODCAR), Giza, Egypt.
De Kraker, M.E.; Wolkewitz, M.; Davey, P.G.;
Conflict of interest Koller, W.; Berger, J.; Nagler, J. et al. (2011).
Clinical impact of antimicrobial resistance in European
The authors declare that they have no conflicts of hospitals: excess mortality and length of hospital stay
interest related to the work done in this manuscript. related to methicillin-resistant Staphylococcus aureus
bloodstream infections. Antimicrobial Agents and
Funding source Chemotherapy. 55(4): 1598-1605.
This study did not receive any fund. Elbeshehy E.K.; Elazzazy A.M., and Aggelis, G.
(2015). Silver nanoparticles synthesis mediated by
Ethical approval
new isolates of Bacillus spp., nanoparticle
Non-applicable, as no in vivo studies were carried out. characterization and their activity against Bean Yellow
Mosaic Virus and human pathogens. Frontiers in
5. References Microbiology. 6: 453.

Bauer A.W.; Kirby W.M.; Sherris J.C. and Turck, Fayaz, A.M.; Balaji, K.; Girilal, M.; Yadav, R.;
M. (1966). Antibiotic susceptibility testing by a Kalaichelvan, P.T. and Venketesan, R. (2010).
standardized single disk method. American Journal of Biogenic synthesis of silver nanoparticles and their
Clinical Pathology. 45(4): 493-496. synergistic effect with antibiotics: a study against

1266
Novel Research in Microbiology Journal, 2021
 Assar et al., 2021

Gram-positive and Gram-negative bacteria. Mohd Yusof, H.; Mohamad, R.; Zaidan, U.H. and
Nanomedicine. 6(1): 103-109. Abdul Rahman, N.A. (2019). Microbial synthesis of
zinc oxide nanoparticles and their potential application
Germain, V.; Li, J.; Ingert, D.; Wang, Z.L. and as an antimicrobial agent and a feed supplement in
Pileni, M.P. (2003). Stacking faults in formation of animal industry: A review. Journal of Animal Science
silver nano-disks. Journal of Physical Chemistry B. and Biotechnology. 10: 57.
107: 8717-8720.
Nayak, B.K.; Chitra, N. and Nanda, A. (2015).
Hallin, M.; Maes, N.; Byl, B.; Jacobs, F.; De Comparative antibiogram analysis of AgNPs
Gheldre, Y. and Struelens, M.J. (2003). Clinical synthesized from two Alternaria spp. with amoxicillin
impact of a PCR assay for identification of antibiotics. Journal of Chemical and Pharmaceutical
Staphylococcus aureus and determination of Research. 7: 727-731.
methicillin resistance directly from blood cultures.
Journal of Clinical Microbiology. 41(8): 3942-3944. Panacek, A.; Kvítek, L.; Prucek, R.; Kolar, M.;
Vecerova, R.; Pizúrova N. et al. (2006). Silver
Karbasian, M.; Atyabim, S.; Siyadat, S.; Momen, S. colloid nanoparticles: synthesis, characterization, and
and Norouzian, D. (2008). Optimizing nanosilver their antibacterial activity. Journal of Physical
formation by Fusarium oxysporum PTCC 5115 Chemistry B .110(33): 16248-16253.
employing response methodology. American Journal
of Agricultural and Biological Sciences. 3: 433-437. Pantidos, N. and Horsfall, L.E. (2014). Biological
Synthesis of Metallic Nanoparticles by Bacteria, Fungi
Li, X.; Xu, H.; Chen, Z.S. and Chen, G. (2011). and Plants. Nanomedicine and Nanotechnology. 5(5):
Biosynthesis of Nanoparticles by Microorganisms 233-242.
and Their Applications. Journal of Nanomaterials. 1-
16. Rajeshkumar, S. and Malarkodi, C. (2014). In Vitro
Antibacterial Activity and Mechanism of Silver
Luteijn, J.M.; Hubben, G.A.; Pechlivanoglou, P.; Nanoparticles against Foodborne Pathogens.
Bonten, M.J. and Postma, M.J. (2011). Diagnostic Bioinorganic Chemistry and Applications. 5881-5890.
accuracy of culture-based and PCR-based detection
tests for methicillin-resistant Staphylococcus aureus: a Sabet, N.S.; Subramaniam, G.; Navaratnam, P. and
meta-analysis. Clinical Microbiology and Infection. Sekaran, S.D. (2007). Detection of mecA and ermA
17(2): 146-154. genes and simultaneous identification of
Staphylococcus aureus using triplex real-time PCR
Makgotlho, P.E.; Kock, M.M.; Hoosen, A.; from Malaysian Staph. aureus strain collections.
Lekalakala, R.; Omar, S.; Dove, M. and Ehlers, International Journal of Antimicrobial Agents. 29(5):
M.M. (2009). Molecular identification and 582-585.
genotyping of MRSA isolates. FEMS Immunology
and Medical Microbiology. 57(2): 104-115. Saifuddin, N.; Wong, C.W. and Nur Yasumirsa,
A.A. (2009). Rapid Biosynthesis of Silver
Marambio-Jones, C. and Hoek, E.M.V. (2010). A Nanoparticles Using Culture Supernatant of Bacteria
review of the antibacterial effects of silver with Microwave Irradiation. E-Journal of Chemistry.
nanomaterials and potential implications for human 6(1): 61-70.
health and the environment. Journal of Nanoparticle
Research. 12(5): 1531-1551. Singh, R.; Wagh, P.; Wadhwani, S.; Gaidhani, S.;
Kumbhar, A.; Bellare J. et al. (2013). Synthesis,
optimization, and characterization of silver

1267
Novel Research in Microbiology Journal, 2021
 Assar et al., 2021

nanoparticles from Acinetobacter calcoaceticus and

their enhanced antibacterial activity when combined
with antibiotics. International Journal of
Nanomedicine. 8: 4277-4290.

Sunkar, S. and Nachiyar, C.V. (2012). Biogenesis of

antibacterial silver nanoparticles using the endophytic
bacterium Bacillus cereus isolated from Garcinia
xanthochymus. Asian Pacific Journal of Tropical
Biomedicine. 2(12): 953-959.

Tubbicke, A.; Hubner, C.; Kramer, A.; Hubner,

N.O. and Flessa, S. (2012). Transmission rates,
screening methods and costs of MRSA-a systematic
literature review related to the prevalence in Germany.
European Journal of Clinical Microbiology and
Infectious Diseases. 31(10): 2497-2511.

Wattal, C. and Oberoi, J.K. (2016). Microbial

identification and automated antibiotic susceptibility
testing directly from positive blood cultures using
MALDI-TOF MS and VITEK 2. European Journal of
Clinical Microbiology and Infectious Diseases. 35(1):
75-82.

Yamal, G.; Sharmila, P.; Rao, K.S. and Pardha, S.

(2013). Inbuilt potential of YEM medium and its
constituents to generate Ag/Ag2O nanoparticles. PLOS
ONE. 8(4): e61750.

1268
Novel Research in Microbiology Journal, 2021