1,2,4,5Department of Biology, Islamic Azad University, Iran
3Department of Biotechnology, University of Maragheh, Iran
Submission: July 25, 2016; Published: August 12, 2016
*Corresponding author: Golestani Emani Bahram, Department of Biology, Urmia Branch, Islamic Azad University, Urmia, Iran.
How to cite this article: Amjady F, Golestani EB, Karimi F, Sevda T, An Investigation of the Effect of Copper Oxide and Silver Nanoparticles on E. Coli Genome by Rapd Molecular Markers. Adv Biotech & Micro. 2016; 1(2): 555559. DOI: 10.19080/AIBM.2016.01.555559
Regarding the bacterial resistance of current antibiotics, many studies have been conducted to evaluate the antimicrobial properties of metal nanoparticles such as silver and copper oxide. This study was carried out to compare the effects of these nanoparticles on the genome of Escherichia coli strain O157: H7 as a model for gram-negative bacteria.
For this purpose, the bacteria were first treated by 30 and 60 μg/ml the nanoparticles and the growth of bacteria was controlled at certain time intervals by spectrophotometry to evaluate the antimicrobial activity. Then the DNA was extracted to investigate the effects of the nanoparticles on the genomes 4 hours after treatment using RAPD-PCR. The bands obtained from electrophoresis of PCR products on agarose gel 2% were analyzed.
The results of the study revealed that silver and copper oxide nanoparticles not only inhibit the growth of bacteria, but also change the genomic DNA sequences and cause genetic differences between control and treated samples. Metal nanoparticles are antibacterial compounds, and copper oxide was more effective than silver on E. coli genome as a model for gram-negative bacteria.
Keywords: Silver and copper oxide nanoparticles; Changes in DNA sequences; Escherichia coli; RAPD polymerase chain reaction (RAPD-PCR)
In general, antibacterial agents are classified into two categories: bactericidal and bacteriostatic (growth inhibitor). However, the widespread and indiscriminate use of anti-bacterial agents has been led to bacterial resistance, which is a serious challenge in this area. Resistance often affects different stages of development such as inheritance to new strains, which requires therapeutic strategies for diseases caused by bacteria . Metal nanoparticles including copper and silver have antimicrobial effect against bacteria, viruses and other microorganisms . Copper nanoparticles, especially inexpensive nanoparticles at the level of micro-electrical applications, have received more attention in recent years and are likely to be the latest discovered antimicrobial agents .
Unlike the usual chemical disinfections, antimicrobial nanomaterials are not expected to manufacture harmful disinfections . Antibacterial effects of metal nanoparticles are because of very small size and high surface area to volume ratio, which allow being in contact directly with microbial membranes and releasing metal ions . Targeted uses of silver and copper oxide nanoparticles with antimicrobial properties require more attention in terms of compatibility with the environment. Considering the fact that copper nanoparticles are lethal to other organisms, for example against crustaceans such as Daphnia magna and Thamnocephalusplatyurus and also Pseudo- kirchneriella subcapitata algae at low concentrations (3.2, 0.18 and <1 mg/l, respectively) reported in 2008 and 2009 [6,7] and thereby a degree of bactericidal of nanoparticles have been shown to aquatic organisms.
It has been also reported that low concentrations of silver ions released from silver nanoparticles are able to haemolysis of red blood cells, in vitro [8,9]. In addition, silver nanoparticles can directly affect the normal functioning of cells in the body, causing dysfunction of the organs . As well, numerous reports and laboratory studies have shown that silver nanoparticles can
induce immune responses . Several experimental researches
have proved that silver nanoparticles cause damages to DNA and
cells as well as result in cancer, oxidative stress and detoxification
of metal [12,13].
Therefore, further investigations are required on
nanoparticles to determine the effective concentrations
of antibacterial and their genotoxicity effects as a suitable
replacement for antibiotics and disinfectants. Since the
antibacterial effects of silver and copper oxide nanoparticles
have not been yet studied on the genomes; so this study was
conducted to evaluate the effect of silver and copper oxide
nanoparticles on the genome of Escherichia coli as a model for
gram-negative bacteria and compare the effect of nanoparticles
on the genome using random amplified polymorphic DNA- PCR
Characteristics of Escherichia coli 0157: H7 (ATCC 25922)
bacteria was identified on eosin methylene blue agar (EMB)
medium. The bacteria were cultured in 5 ml of the Brain Heart
Infusion (BHI) broth, and were left overnight in a shaking
incubator at 37°C at rpm 200. The growth rate of bacteria was
controlled by measuring optical density (OD) of medium at a
wavelength of 600 nm .
Copper oxide and silver nanoparticles less than 20nm in
diameter were synthesized by the Nanotechnology South Korea.
Characterization and analysis of the nanoparticles by electron
microscopy have been presented in (Figure 1). Phosphate buffer
saline (pH 7.4) was used as a solvent for preparing 30 and 60μg/
ml concentrations of the nanoparticles, separately in tubes. The
tubes were placed in a shaking incubator at 37°C at 200rpm
and their OD was measured at 600nm with intervals of 2, 4 and
24 hours for the bacteria treated by nanoparticles to measure
DNAs of treated and control bacteria were extracted using
DNA extraction kit (ExirAzma Co.) in accordance with the kit
instructions, and then the quantity and quality were analyzed
by spectrophotometry and electrophoresis on agarose gel 1%.
RAPD- PCR molecular marker method was used to examine the
effect of nanoparticles on bacterial genome. Initially, RAPD 10
bases-primers (Cinnagene Co.) were prepared to perform RAPDPCR
method. Sequences and characterization of primers have
been shown in (Table 1).
Ingredients listed below at concentrations of 25ml volume
were prepared for PCR reaction to amplify samples; 1μl primer,
2.5μl (10 x) PCR buffer, 3μl MgCl2, 1μl dNTP mix, 1μl of extracted
DNA samples and 0.3μl of DNA Taq polymerase that was reached
to the volume of 25ml by 16.2μl of deionized distilled water.
The mixture was placed in a thermocycler (Corbett research,
Australia) with the following schedule: the initial template
DNA denaturation at 95°C for 5 minutes, followed by 40 cycles
of PCR reactions, so that 95°C for 35 seconds for denaturation
of template DNA strands, 30°C for 45 seconds to attach the
primers to the template strand, 72°C for 45 seconds for the
polymerization of a new strand from template strand. 7 minutes
were required to complete the polymerization of incomplete
strands. These compositions and the temperature profiles after
optimizing the PCR conditions were used for all 14 primers.
After completion of the PCR reaction, electrophoresis was
done for 10μl of PCR products on agarose gel 2% (the size of
14×26cm) containing red safe in TBE buffer (1x) for 4 hours
with a voltage of 120 volt to detect appeared bands; DNA ladder
marker with 100bp was used to determine the product size
and the images of gel were taken using imaging system (Uvitec,
The bands resulting from analyzed RAPD were scored
based on the presence or absence, respectively, as one and
zero. The data was then entered into the software based on
molecular weight; the similarity matrix was calculated by Dic
and dendrogram were derived by UPGMA method in NTSYS-PC
The results of experiments on antimicrobial activities of
copper oxide and silver nanoparticles against Escherichia coli
bacteria have been presented in (Figure 2), demonstrating that
the growth of bacteria was clearly stopped after treatment by
nanoparticles in intervals of 2 and 4 hours and they had only
minor growth after 24 hours.
Electrophoretic bands obtained from amplification of 14
primers by the RAPD- PCR have been shown in (Figure 3) for
bacteria treated by copper oxide and silver nanoparticles.
The bands obtained from analyzed RAPD were scored based
on the presence or absence, respectively, as one and zero. The
conclusion was based on the difference in the bands formed by
each primer, for control and treated samples (Table 2) as can be
seen in (Table 2), totally 53 bands were produced for bacteria
treated with copper oxide nanoparticles from 14 primers
that 41 bands were different between the control and treated
samples. Collectively 73 bands were also appeared for bacteria
treated with silver nanoparticles which 60 bands were different
between the control and treated samples.
(Table 3) shows the results of NTSYS-PC software in order to
compare genetic variations between control and treated samples.
The dendrogram was drawn by UPGMA in NTSYS-PC
software to compare genetic variations between control and
samples treated with nanoparticles. As seen in (Figure 4),
control and samples treated with copper oxide nanoparticles
have been located in two separate main branches, suggesting
a genetic difference; but the control and first treatment of
silver nanoparticles in a branch, and the second treatment in a
separate main branch, reflecting the impact of nanoparticles at
The development of severe bacterial resistance to antibiotics
is a major health problem. In this regard, the nanoparticles are
considered new antimicrobial agents . Various industries
and human daily life have been changed with the advent of
nanotechnology. Antimicrobial potential of nanoparticles has
attracted the attention of researchers and industrialists, which
nanoparticles can be used as an alternative to antibacterial
agents and antibiotics, as it is predicted that bacteria cannot
become resistant to nanoparticles because nanoparticles can
be effective on different parts and various enzymes, which this
issue is confirmed by research of Sondi et al.  in 2004 on
the antimicrobial activity of silver nanoparticles. They argued
that antimicrobial activity of silver is carried out by blocking
the electron transport system, changing the bacterial membrane
function and inhibit the DNA replication. Silver ions are known
to particularly inhibit thiol group-containing enzymes and
proteins and thereby this mechanism plays an essential role in
the antimicrobial activity of silver ions, although other cellular
factors such as hydrogen bonds may also be involved .
New Delhi metallo-beta-lactamase-1 (NDM-1) is an enzyme
that makes bacteria resistant to a broad range of beta-lactam
antibiotics.Most isolates with NDM-1 enzyme are resistant to
all standard intravenous antibiotics for treatment of severe
infections . DNA gyrase enzyme in E.coli participates in
several important processes, and thus is physiological target for
a series of antibiotics. E.coli mutants resistant to the two classes
of drugs have provided important evidences about subunit
structure of the enzyme.
Drug resistance is controlled by two groups of genes
(gyr A and gyr B) that are structural genes for subunits of the
enzyme [18,19]. Therefore, the nanoparticles can be used for
antimicrobial activities.But both advantages and disadvantages
need to be considered in the host cell. Nanoparticles arenot toxic
to cells in the body at low concentrations.In this study, certain
concentrations of silver and copper oxide nanoparticles were
used for bacterial treatment to find out antimicrobial properties.
The results of the present study (Figure 1) showed that the
nanoparticles with diameters less than 20nm in doses of 30 and
60μg/ml had relatively good antimicrobial effects so that were
able to almost inhibit the growth of all bacteria in the samples.
According to numerous studies that have demonstrated the
effect of silver and copper oxide nanoparticles as antimicrobial
agents [16,20-25], the main purpose of this research was to
evaluate and compare the effect of nanoparticles on bacterial
genome at the lowest effective dose.Reports have been also
presented based on the nanoparticles effects on bacterial
genome which can induce DNA single-strand breakage and affect
gene expression .
Li et al.  in 2012 during a study stated that silver
nanoparticles are imported into bacterial cells and influence
on the DNA twisting, thus inhibit the replication and cell proliferation. The silver nanoparticles are combined with the
thiol groups in respiratory enzymes and inhibit respiration
process in bacterial cells [27,28] or as expressed in previous
studies, metal oxide nanoparticles may interfere with the
transcription and translation .
This study was also conducted to investigate the effects
of silver and copper oxide nanoparticles on the genome of
Escherichia coli strain O157: H7 as a model for gram-negative
bacteria. In this regard, based on the RAPD-PCR reaction with
14 primers, the presence or absence of bands in the gel images
(Figure 3) suggest changing the DNA sequence by silver and
copper oxide nanoparticles. A large number of primers failed
to detect target sequences, and therefore the related segments
were unable to replicate and we have seen the absence of bands
on the agarose gel.
The difference among the bands observed in the treated and
control groups of bacteria suggests that the target sequences
of primers have been changes in the treated bacteria that make
a difference in binding of primers and PCR amplification. The
genomic sequence variations could possibly be in the process of
It can be concluded that a change in the base pairing
properties could be one of the possible causes of DNA sequence
variations due to treatments of bacteria by silver and copper
oxide nanoparticles which during the replication can lead
to change the sequences in daughter strands. Also, silver
nanoparticles could possibly cause dysfunction in DNA pol
enzyme and are able to target the molecular mechanisms of
replication accuracy which is involved in the synthesis of new
strands based on the structure of Watson and Crick, changing
the sequence of daughter strands .
Variations observed in DNA sequence in this study could
also be a factor for growth inhibition and cell cycle through the
occurrence of mutations, followed by gene expression changes
associated with growth and cell cycle control . Copper oxide
and silver nanoparticles inside the cells can release ions of copper
oxide [30,31] and silver, which react with DNA phosphorus and
then disable the replication.
Silver ions increase the level of ROS, react with the sulfurcontaining
proteins and inhibit the respiratory enzymes,
resulting in cell death [31,32]. The energetic ions of copper
cations with moving easily among the lipid layers are trapped by
the cells which produce a specific reaction of oxygen, penetration
of lipid peroxidation and protein oxidation.
Cell wall components are responsible for binding with copper
nanoparticles. Amine and carboxyl groups of peptidoglycan
participate in copper process and cell wall damages .
Accordingly, given that growing and replicating bacteria in the
present research have been treated with silver and copper oxide
nanoparticles, it could be argued that these nanoparticles most
likely can create disruption in replication as well as in repair mechanisms to cause multiple mutations in DNA sequences.
In accordance with the results of (Figure 1) there are
significant differences between treatment and control samples
and in fact bacteria were separated into two distinct strains in
terms of genome. Based on dendrogram, samples of control and
treated with copper oxide nanoparticles by being in separate
categories demonstrate great genetic distances. But the control
and first treatment of silver nanoparticle were placed in single
branch, and the second treatment was subjected to a separate
main branch, reflecting the impact of nanoparticles on bacterial
genome at high concentrations. Therefore, according to previous
studies and the current research, it can be concluded that the
nanoparticles can reduce the expression of genes involved in cell
cycle control by creating mutations in their sequences, and thus
reduce the growth of bacteria.
The results of this study and similar findings indicate the
proper efficacy of nanoparticles as antibacterial compounds, but
copper oxide nanoparticles compared with silver nanoparticles
was more effective on E. coli genome as the model for gramnegative
bacteria and since the copper nanoparticles are less
expensive, thus is cost-effective as antibacterial agents. But
because the nanoparticles can bind with DNA, so in the long term
can make hard mutants and have adverse effects on eukaryotic
host cells and it is impossible to be tested in a short time. But it is
suggested to be further investigated in future works on the effects
of nanoparticles in eukaryotic cells, which the nanoparticles can
be used in various industries with greater certainty.
The authors hereby express their appreciation and gratitude
to Biotechnology Department, University of Maragheh, Iran, due
to sincere cooperation in providing primers and nanoparticles
used in this study.