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Nanomed. J., 2(4): 291-298, Autumn 2015
291
Synthesis of silver nanoparticles and its synergistic effects in combination
with imipenem and two biocides against biofilm producing Acinetobacter
baumannii
1
S. Hendiani; 1*
A. Ahya Abdi; 1
P. Mohammadi; 2
Sh. Kharrazi
1
Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
2
Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University
of Medical Sciences, Tehran, Iran
ABSTRACT:
Objectives: Biofilms are communities of bacteria attached to surfaces through an external polymeric substances matrix.
In the meantime, Acinetobacter baumanniiis the predominant species related to nosocomial infections. In the present
study, the effect of silver nanoparticles alone and in combination with biocides and imipenem against planktonic and
biofilms of A. baumannii was assessed.
Materials and Methods: Minimum inhibitory concentrations (MICs) of 75 planktonic isolates of A. baumannii were
determined by using the microdilution method as described via clinical and laboratory standards institute (CLSI).
Among all strains, 10 isolates which formed strong biofilms were selected and exposed to silver nanoparticles alone and
in combination with imipenem, bismuth ethandithiol (BisEDT) and bismuth propanedithiol (BisPDT) to determine
minimum biofilm inhibitory concentrations (MBIC). Subsequently, minimum biofilm eradication concentrations
(MBECs) of silver nanoparticles alone and in combination with imipenem against mature biofilm of the isolates were
evaluated.
Results: Results showed that 29.3% of isolates were susceptible to silver nanoparticles and could inhibit the growth and
eradicate biofilms produced by the isolates. For this reason, “FIC, “FBIC and “FBEC d” 0.05 were reported which
shows synergism between silver nanoparticles and imipenem against not only planktonic cells but also inhibition and
eradication of biofilms. The results of “FBIC >2 indicated to antagonistic impacts between silver nanoparticles and
BisEDT/BisPDT against biofilms.
Conclusion: It can be concluded that silver nanoparticles alone can inhibit biofilm formation but in combination with
imipenem are more effective against A. baumannii in planktonic and biofilm forms.
Keywords: Acinetobacter baumannii, Biofilm,Bismuth ethandithiol (BisEDT),Bismuth propanedithiol (BisPDT), Imipenem,
Silver nanoparticles (AgNPs)
Nanomed. J., 2(3): 291-298, Autumn 2015
DOI: 10.7508/nmj. 2015.04.007
*Corresponding Author Email: abdialya@alzahra.ac.ir
Tel: (+98) 21-88044040
Note. This manuscript was submitted on June 20, 2015;
approved on August 7, 2015
Received; 20 June 2015 Accepted; 7 August 2015
INTRODUCTION
Silver is a metal known for its broad-spectrum of
antimicrobial activities againstGram-positive and Gram-
negative bacteria, fungi, protozoa and certain viruses.
In addition, it was reported that using silver
compounds in medical products such as dental fillings,
catheters, bandages and implants decreases the chance
of bacterial infections (1). The emergence and spread
of antibiotic resistant pathogen is a distressing concern
in clinical practices. There is a need of a cheap broad
spectrum agent that can be used against variety of
pathogens. The silver nanoparticles (AgNPs) have
been found to be effective against various viruses and
bacteria (2). During the last decades, introducing of
AgNPs has exhibited enhanced antimicrobial properties
(3-5).AgNPs anchor to the cell wall and penetrate into
of the Gram-negative bacteria cell membrane, increasing
the cell permeability, leading to an uncontrolled
transport through the cytoplasmic membrane and
finally cell death (4). Furthermore, interaction ofAgNPs
Nanomed. J., 2(4): 291-298, Autumn 2015
292
S. Hendiani et al.
with DNA of bacteria inhibit DNA replication, and
ultimately inducing cell death (6). It has been also
proposed that the antibacterial mechanism of AgNPs
is correlated to the formation of free radicals and later
on, the free radical-induced membrane damages (7).
The properties of nanoparticles can be affected by their
size and shape, chemistry of the affected surface,
solubility and aggregation of particles (8). Moreover,
AgNPs as an effective agent against broad spectrum
of Gram-negative and Gram-positive bacteria was
reported through the preventing of biofilm formation
(3). It was investigated that silver chelates or silver
ions are more effective as therapeutic agents. To
examine their mode of action and therapeutic activities
against Gram positive bacteria is very difficult with
AgNPs alone, although increasing percentage of
effectiveness are the same for ampicillin in combination
with AgNPs against Gram positive and Gram negative
bacteria. It seems ampicillin molecules come into
contact withAgNPs, wherein silvernano core which is
surrounded by ampicillin molecules(9). Likewise, it was
observed that the antibacterial activity of penicillin G,
amoxicillin, erythromycin, clindamycin, and
vancomycin increased in the presence of AgNPs
against Staphylococcus aureus and Escherichia coli
(10). There are evidences of antibacterial effects of
AgNPs and synergetic capacity on planktonic cells
and biofilm structures. These results suggest that
AgNPs have potential activity to use as an adjuvant
components to treat the infectious diseases (11).
Acinetobacterbaumanniiis a significant worldwide
nosocomial pathogen with ability to extend
antimicrobial resistance and cause nosocomial
outbreaks of infection (12). This organism commonly
causes infections related to medical devices, e.g.,
vascular catheters, cerebrospinal fluid shunts or Foley
catheters (13). Biofilm formation is a well-known
pathogenic mechanism in Acinetobacter infections
(14). The ability of A. baumannii to form biofilms could
explicate its antibiotic resistance and survival
properties (15). Bioûlm formation occurs as a sequence
of procedures: microbial surface attachment, cell
proliferation, matrix production and detachment (16).
Bioûlms create an environment that increases
antimicrobial resistance (17). Imipenem is a β-
lactamases-resistant carbapenem with broad and high
antibacterial activities against bacteria. This antibiotic
has superior affinity for the PBP2 of Gram-negative
bacteria, thus prevents cell division and resulting the
cell death (18).
Bismuththiols (BTs) are a new class of biocides with
antibiofilm activity against bacteria. The thiol
component acts as a lipophilic carrier that increases
bismuth uptake by the bacteria, therefore enhances
the effects of bismuth up to 1000-fold. Inside the cells,
bismuth acts as a metabolic poison, resulting in the
growth inhibition and the cell death. At sub-inhibitory
concentrations, BTs inactivate bacterial respiratory
enzymes, suppress exopolysaccharide expression and
subsequently inhibit biofilm formation of bacteria (19).
Due to the clinical importance of A. baumannii as well
its ability to form biofilm, inhibition and eradication of
this structure was investigated in the presence of
AgNPs alone and in combination with antimicrobial
agents.
MATERIALS AND METHODS
Bacterial isolates
In this study, 65 isolates of A. baumannii where
collected from previous investigations (55 isolates from
burn wounds , Burn hospital, Tehran, Iran and 10
isolates from urinary catheters,Army hospital, Tehran,
Iran) were used. Frozen stocks were stored in skim milk
(Merck, Germany) containing 15% glycerol at -70 °C.
Isolates were transferred from the stock cultures into
tryptic soy agar [TSA, (Merck, Germany)] and were
aerobically incubated at 37 °C for 24 h.
All tests were carried out in duplicate. Quality control
as antibiotic susceptibility test were Pseudomonas
aeroginosaATCC 27853 and E.coli ATCC 27922.
Antimicrobial agents
The AgNPs were prepared via chemical reduction
of silver ions (AgNO3
). For this reason, nanoparticles
were prepared by mixing of an aqueous solutions of
Ag ions (0.5 mL, 0.1 mM) with aqueous solution of
sodium citrate (12 mL, 0.1 mM). Then, aqueous solution
of NaBH4
(0.5 mL, 0.1 M) was added into the above
solution to form a stable colloidal solution of AgNPs.
Imipenem (Exir, Iran) stock solution were prepared in
0.01 Mphosphate buffer and diluted as described by
CLSI protocol.
Bismuth ethandithiol (BisEDT) and Bismuth
propanedithiol (BisPDT) were two biocides which was
used in this study. For this reason, 4.58 mg of bismuth
nitrate (BN) was dissolved in 1 mL of 1,2-propandiol.
Either 100 µL of 1,2-ethanedithiol [EDT, (Merck,
Germany)] or 100 µL of 1,2-propanedithiol [PDT,
(Merck, Germany)] was dissolved in 900 µl of 1,2-
Nanomed. J., 2(4): 291-298, Autumn 2015
293
propandiol. Then, 1000 µg/mL working stock solution
of each biocide was prepared by adding 50 µl of BN,
5.8 µlofHCl(12.1 M)and4.2µlofEDT orPDT to940µl
of 1,2-propandiol (20).
MIC determination of silver nanoparticles on
planktonic bacteria
To determine the minimum inhibitory concentration
of AgNPs on planktonic bacteria, serial dilutions of
AgNPs were prepared by diluting 15 times of 100 ppm
solution of AgNPs to reach to the half
concentration.Suspension of cells in Muller Hinton
broth was adjusted to 0.5 McFarland tubeand diluted
1:100 in Muller Hinton broth medium. Then, 100 µl of
diluted AgNPs was added to each row of microtiter
plate followed by addition of 100 µL of each isolates to
the wells. Finally, microtiter plates were incubated at
37°C for 24 h (21).Assessment of synergetic effect of
AgNPs in combination with imipenem on planktonic
bacteria was determined by using checkerboard
microdilution technique. Stock solutions of
imipenemandAgNPsweredilutedand 50µLofimipenem
dilutions (0.25-1024 µg/mL) were added to each row of
microtiter plate.Afterward, 50 µLofAgNPs (0.003-100
ppm) were added to each column of microtiter plates.
Then, 100 µL of each bacterium was added to each
well. Plates were incubated at 37°C for 24 h (22).After
that, the fractional inhibitory concentration (“FIC) for
both antimicrobials was calculated according to
European Committee for Antimicrobial Susceptibility
Testing (EUCAST) of the European Society of Clinical
Microbiology and Infectious Diseases (2000):
“FIC d”0.5 is a sign of synergism, >0.5-1 sign of
addition, >1 to 2 indifference and e”2 is a sign of
antagonism.
MIC determination of silver nanoparticles against
biofilm
For determination of minimum biofilm inhibitory
concentration (MBIC) and minimum biofilm eradication
concentration (MBEC) of nanoparticles, 10 isolates out
of 75 isolates of A. baumanniiwere chosen. The
criterion for theselection was the ability of these strains
to form strong biofilms as described in previously
study(23).
To determine MBIC of silver nanoparticles,serially
diluted nanoparticles were prepared in TSB.
Subsequently, microtiter plates were filled and
incubated by AgNPs and suspension of cells,
respectively as described for planktonic cells.
Subsequently, plates were emptied and washed 5 times
with phosphate buffer saline (PBS) and were left to
dry. This step was followed by adding 200 µL of 0.2 %
crystal violet. After 5 minutes; the plates was washed
for 5 times and destained by using 33 % (v/v) glacial
acetic acid. Next, 100 µL of each well contents were
transferred to the wells of a new microtiter plate and
the plates were read by an ELISA plate reader (24).
The efficacy of biocides was calculated according to
the following equation.
Where B denotes the average of absorbance for blank
wells, C denotes the average of absorbance for control
wells and T denotes the average of absorbance for
treated wells (25).
To determine MBIC of AgNPs in combination with
imipenem, Bismuth-ethandithiol(BisEDT) and Bismuth-
propanedithiol (BisPDT) stock solutions of each
antimicrobial were prepared in tryptic soy broth [TSB,
(imipenem0.125-1024µg/mL,BisEDTandBisPDT0.09-
1000 µg/mL)] and synergetic effect of AgNPs as well
fractional biofilminhibitoryconcentration (“FBIC) were
determined.
To determine minimum biofilm eradication
concentration (MBEC), each microtiter plate was filled
with given concentration of isolates and incubated for
24 h at 37°C to form biofilm. Next, plates were emptied
and washed 5 times to remove non-biofilm forming cells
(25). To determine of MBEC ofAgNPs,serially diluted
concentrations of AgNPs were added to each well and
incubated at 37 ° C for 20 minutes. Then plates were
emptied and filledagain and repeated to get the final
time of 1h.Afterward, plates were emptied and washed
5 times and followed by adding up neutralizer for 5
minutes (25). The sodium salt of XTT (2,3-Bis-(2-
methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
carboxanilide)(Sigma) was dissolved in phosphate
buffer saline to the concentration of 1 mg/mL. In
addition, menadione (Sigma) was dissolved in acetone
to reach to 1 mM concentration. The XTT/menadione
Nanomed. J., 2(4): 291-298, Autumn 2015
294
Effect of AgNPs against Acinetobacter biofilms
reagent must be prepared fresh prior to each assay and
contained 12.5 parts XTT/1 part menadione. Then, 100
µL XTT-menadione were added to each well and
incubated at 37°C in a shaker incubator at 150 r.p.m for
1h. Finally, absorbance at 505 nm were read by plate
ELISA reader (26).The efficacy of biocide was
calculated as described above.
To determine of MBEC ofAgNPs in combination with
imipenem, diluted antimicrobials were added to plates
via checkerboard method and fractional biofilm
eradication concentration (“FBEC) were then
determined.
Scanning electron microscopy was applied to determine
effectiveness of antimicrobial agents to eradicate
b i o f i l m . G l a s s s l i d e s w i t h d i m e n s i o n o f 1 × 1 c m
2
were
placed in the wells of a 12-well sterile microtiter plate.
The steps was followed by adding 3 mL of bacterial
suspension and mediumin the first well, 1.5 mL bacterial
suspension and 1.5 mL AgNPs in the second well and
1.5 mL bacterial suspension and 1.5 mLAgNPs and
imipenem into the third well. The plate was incubated
at 37°C for 24 h. After that, slides were washed by
sterile distilled water and placed in Glutaraldehyde 4%
for 2 h to fix.
Then, to dehydrate all slides, they were put for 15 min
in the serial concentrations of 40 %, 60 %, 80 %, 90 %
and 100 % of absolute ethanol (Merck, Germany),
respectively. Finally, biofilms on the surface of the
glasses were lyophilized. Electron microscopic
micrographs were taken using Phillips XL 30.
Statistical analysis
Results were analyzed with SPSS software using
one way ANOVA. All experiments were performed in
triplicate. P values of d” 0.05 were regarded as
significant.
RESULTS
In this study, MIC of AgNPs against planktonic
bacteria was determined. Only 22 isolates (29.3%)
showed susceptibility to 100 ppm and remaining
isolates were resistant to all AgNPs concentrations.
Also, MBIC of AgNPs alone and in combination with
imipenem, BisEDT and BisPDT was determined (Fig.
1). The concentrations of AgNPs, imipenem, BisEDT
and BisPDT for all isolates has been shown in Table 1.
As showed in scanning electron microscopy
micrographs, AgNPs alone could reduce biofilm of A.
baumannii isolates, but in combination with imipenem,
biofilm producing isolates could not attach to the
surface of glass and form the biofilm (Fig. 2).
In the other hand, in comparison with AgNPs alone,
AgNPs in combination with imipenem and biocides
could inhibit biofilm formation of A. baumannii at lower
concentrations.
Moreover, results indicated synergistic activity of
AgNPs and imipenem. However, antagonistic impact
of AgNPs with BisEDT and BisPDT was clearly
observed.
MBEC of AgNPs alone and in combination with
imipenem were also determined and presented in Fig.
3. Since generation time of Acinetobacter isolates was
reported 20 minutes in previous(27), it seems after
induction of cell division, AgNPs can inhibit this
process resulting in cell death.
As it was shown in the Table 1, synergistic impact
between AgNPs and imipenem to eradicate biofilms
was observed.
Furthermore, statistical analysis showed significant
differences between AgNPs alone and in combination
with antimicrobials data with P values of d” 0.05.
Finally, the combination of AgNPs and imipenem at
lower concentrations could be inhibited biofilm
formation of A.baumannii.
Fig. 1. MBIC of AgNPs alone and in combination with
imipenem, BisEDT and BisPDT was shown
Nanomed. J., 2(4): 291-298, Autumn 2015
295
Table 1. Concentrations (µg/mL) of AgNPs, imipenem.BisEDT and BisPDT of MBIC were presented
isolate 1 2 3 4 5 6 7 8 9 10
AgNPs AgNPs 0.8 6.5 1.6 6.5 6.5 6.5 6.5 0.8 0.8 6.5
AgNPs +
IM1
AgNPs 0.0003 0.8 0.4 0.0015 0.0003 0.0031 0.0003 0.2 0.0003 0.0003
IM 0.25 32 32 8 0.125 32 1 16 16 16
AgNPs +
BisEDT
AgNPs 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.4
BisEDT 16 8 32 32 32 16 16 32 16 16
AgNPs +
BisPDT
AgNPs 0.2 0.4 0.2 0.2 0.2 0.2 0.2 0.4 0.2 0.2
BisPDT 16 16 32 16 16 16 16 16 16 32
1
IM = imipenem
Fig. 2. Scanning electron micrographs of (a) and (b) positive control with different magnification, (c) and (d) treated
the isolates with AgNPs alone with different magnification, (e) mixed AgNPs and imipenem treatment against the
same isolate number 10
Nanomed. J., 2(4): 291-298, Autumn 2015
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S. Hendiani et al.
Fig. 3. MBEC of AgNPs alone and in combination with
imipenem
DISSCUSION
It was suggested that the positively charged AgNPs
can attach to the lipopolysaccharides of Gram-negative
bacteria and to be effective via metal reduction(29),
However, Hong et al. reported that negativelycharged
AgNPs can be electrostatically repulsed from the
negatively charged (-20 mV) surfaces of bacteria (30).
This feature could be attributed to the long time exposure
(for example 24 hours) of nanoparticles in which
resistance mechanisms like efflux systems could be
deactivated the antimicrobial activity of AgNPs. In this
study, synergistic effect of AgNPs in combination with
imipenem(“FICd” 0.05) against allisolates was observed.
According to results of MBIC, it could be suggested
that the excretion of adhesive substances such as
polysaccharides and proteins is crucial factor for the
initial attachment of organisms as well to hold the
bacteria together. It was reported that the AgNPs might
be involved in preventing these adhesive substances
and thereby resulting in prevention of biofilm
formation(31).
Since A. baumannii biofilm matrix comprises of
extracellular DNA (eDNA),itseems that reaction between
positively chargedAgNPs and negatively charged eDNA
play a major role in the inhibitionof biofilm formation in
isolates.
BTs are the new classes of anti-biofilm agents that has
been shown to have remarkable activity against a broad
spectrum of biofilm producing organisms through
inhibiting the slim expression in both Gram-positive
and Gram-negative bacteria at non-toxic
concentrations (32). In this study, it was assumed
that the combination of AgNPs and BisEDT or
BisPDT have a synergistic impact. Although both
biocides are exopolysaccharide inhibitors but they
have antagonistic activity when used together.
The synergism between AgNPs and imipenem could
be due to the damage of the matrix and consequently
improved penetration of antibiotic. Furthermore,
antibiotic molecules have many active sites such as
hydroxyl and amine groups which can interact
withAgNPs and act as a chelating agent.
Moreover,imipenem lyses the cell wall of bacteria
leading to increase in penetration of AgNPs into the
cells.
Thiols can chelate bismuth and other metals. It seems
in the presence of AgNPs, thiols preferably bond to
silverionsleading to easier penetration of Ag into the
cells. Therefore, lower concentrations ofAgNPs when
mixed with thiols, can inhibit biofilm formation.
However, higher concentrations of thiol would be
required when there is antagonism (“FBIC > 2)
between AgNPs and thiols.
CONCLUSION
This study demonstrated a synergistic effect of
imipenem andAgNPs against planktonic cells as well
biofilms, making it a promising candidate for a new
treatment of infections caused by MDR A. baumannii
strains. Further investigation should be carried out
to be sure of effectiveness of this nanoparticle in
vivo experiments and assay probable cytotoxicity of
this material.
ACKNOWLEDGEMENTS
The authors wish to thank Vice Chancellor of
Alzahra University and Iran nanotechnology initiative
council for the financial support and Burn and Army
hospitals for providing the bacterial isolates.
CONFLICTOFINTEREST
No conflict of interest.
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How to cite this article:
Hendiani S, Ahya Abdi A, Mohammadi P, Kharrazi Sh. Synthesis of silver nanoparticles and its Synergistic effects in combination with
imipenem and two biocides against biofilm producing Acinetobacterbaumannii. Nanomed. J., 2015; 2(4): 291-298.

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Synthesis of silver nanoparticles and its synergistic effects in combination with imipenem and two biocides against biofilm producing Acinetobacter baumannii

  • 1. Nanomed. J., 2(4): 291-298, Autumn 2015 291 Synthesis of silver nanoparticles and its synergistic effects in combination with imipenem and two biocides against biofilm producing Acinetobacter baumannii 1 S. Hendiani; 1* A. Ahya Abdi; 1 P. Mohammadi; 2 Sh. Kharrazi 1 Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran 2 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran ABSTRACT: Objectives: Biofilms are communities of bacteria attached to surfaces through an external polymeric substances matrix. In the meantime, Acinetobacter baumanniiis the predominant species related to nosocomial infections. In the present study, the effect of silver nanoparticles alone and in combination with biocides and imipenem against planktonic and biofilms of A. baumannii was assessed. Materials and Methods: Minimum inhibitory concentrations (MICs) of 75 planktonic isolates of A. baumannii were determined by using the microdilution method as described via clinical and laboratory standards institute (CLSI). Among all strains, 10 isolates which formed strong biofilms were selected and exposed to silver nanoparticles alone and in combination with imipenem, bismuth ethandithiol (BisEDT) and bismuth propanedithiol (BisPDT) to determine minimum biofilm inhibitory concentrations (MBIC). Subsequently, minimum biofilm eradication concentrations (MBECs) of silver nanoparticles alone and in combination with imipenem against mature biofilm of the isolates were evaluated. Results: Results showed that 29.3% of isolates were susceptible to silver nanoparticles and could inhibit the growth and eradicate biofilms produced by the isolates. For this reason, “FIC, “FBIC and “FBEC d” 0.05 were reported which shows synergism between silver nanoparticles and imipenem against not only planktonic cells but also inhibition and eradication of biofilms. The results of “FBIC >2 indicated to antagonistic impacts between silver nanoparticles and BisEDT/BisPDT against biofilms. Conclusion: It can be concluded that silver nanoparticles alone can inhibit biofilm formation but in combination with imipenem are more effective against A. baumannii in planktonic and biofilm forms. Keywords: Acinetobacter baumannii, Biofilm,Bismuth ethandithiol (BisEDT),Bismuth propanedithiol (BisPDT), Imipenem, Silver nanoparticles (AgNPs) Nanomed. J., 2(3): 291-298, Autumn 2015 DOI: 10.7508/nmj. 2015.04.007 *Corresponding Author Email: abdialya@alzahra.ac.ir Tel: (+98) 21-88044040 Note. This manuscript was submitted on June 20, 2015; approved on August 7, 2015 Received; 20 June 2015 Accepted; 7 August 2015 INTRODUCTION Silver is a metal known for its broad-spectrum of antimicrobial activities againstGram-positive and Gram- negative bacteria, fungi, protozoa and certain viruses. In addition, it was reported that using silver compounds in medical products such as dental fillings, catheters, bandages and implants decreases the chance of bacterial infections (1). The emergence and spread of antibiotic resistant pathogen is a distressing concern in clinical practices. There is a need of a cheap broad spectrum agent that can be used against variety of pathogens. The silver nanoparticles (AgNPs) have been found to be effective against various viruses and bacteria (2). During the last decades, introducing of AgNPs has exhibited enhanced antimicrobial properties (3-5).AgNPs anchor to the cell wall and penetrate into of the Gram-negative bacteria cell membrane, increasing the cell permeability, leading to an uncontrolled transport through the cytoplasmic membrane and finally cell death (4). Furthermore, interaction ofAgNPs
  • 2. Nanomed. J., 2(4): 291-298, Autumn 2015 292 S. Hendiani et al. with DNA of bacteria inhibit DNA replication, and ultimately inducing cell death (6). It has been also proposed that the antibacterial mechanism of AgNPs is correlated to the formation of free radicals and later on, the free radical-induced membrane damages (7). The properties of nanoparticles can be affected by their size and shape, chemistry of the affected surface, solubility and aggregation of particles (8). Moreover, AgNPs as an effective agent against broad spectrum of Gram-negative and Gram-positive bacteria was reported through the preventing of biofilm formation (3). It was investigated that silver chelates or silver ions are more effective as therapeutic agents. To examine their mode of action and therapeutic activities against Gram positive bacteria is very difficult with AgNPs alone, although increasing percentage of effectiveness are the same for ampicillin in combination with AgNPs against Gram positive and Gram negative bacteria. It seems ampicillin molecules come into contact withAgNPs, wherein silvernano core which is surrounded by ampicillin molecules(9). Likewise, it was observed that the antibacterial activity of penicillin G, amoxicillin, erythromycin, clindamycin, and vancomycin increased in the presence of AgNPs against Staphylococcus aureus and Escherichia coli (10). There are evidences of antibacterial effects of AgNPs and synergetic capacity on planktonic cells and biofilm structures. These results suggest that AgNPs have potential activity to use as an adjuvant components to treat the infectious diseases (11). Acinetobacterbaumanniiis a significant worldwide nosocomial pathogen with ability to extend antimicrobial resistance and cause nosocomial outbreaks of infection (12). This organism commonly causes infections related to medical devices, e.g., vascular catheters, cerebrospinal fluid shunts or Foley catheters (13). Biofilm formation is a well-known pathogenic mechanism in Acinetobacter infections (14). The ability of A. baumannii to form biofilms could explicate its antibiotic resistance and survival properties (15). Bioûlm formation occurs as a sequence of procedures: microbial surface attachment, cell proliferation, matrix production and detachment (16). Bioûlms create an environment that increases antimicrobial resistance (17). Imipenem is a β- lactamases-resistant carbapenem with broad and high antibacterial activities against bacteria. This antibiotic has superior affinity for the PBP2 of Gram-negative bacteria, thus prevents cell division and resulting the cell death (18). Bismuththiols (BTs) are a new class of biocides with antibiofilm activity against bacteria. The thiol component acts as a lipophilic carrier that increases bismuth uptake by the bacteria, therefore enhances the effects of bismuth up to 1000-fold. Inside the cells, bismuth acts as a metabolic poison, resulting in the growth inhibition and the cell death. At sub-inhibitory concentrations, BTs inactivate bacterial respiratory enzymes, suppress exopolysaccharide expression and subsequently inhibit biofilm formation of bacteria (19). Due to the clinical importance of A. baumannii as well its ability to form biofilm, inhibition and eradication of this structure was investigated in the presence of AgNPs alone and in combination with antimicrobial agents. MATERIALS AND METHODS Bacterial isolates In this study, 65 isolates of A. baumannii where collected from previous investigations (55 isolates from burn wounds , Burn hospital, Tehran, Iran and 10 isolates from urinary catheters,Army hospital, Tehran, Iran) were used. Frozen stocks were stored in skim milk (Merck, Germany) containing 15% glycerol at -70 °C. Isolates were transferred from the stock cultures into tryptic soy agar [TSA, (Merck, Germany)] and were aerobically incubated at 37 °C for 24 h. All tests were carried out in duplicate. Quality control as antibiotic susceptibility test were Pseudomonas aeroginosaATCC 27853 and E.coli ATCC 27922. Antimicrobial agents The AgNPs were prepared via chemical reduction of silver ions (AgNO3 ). For this reason, nanoparticles were prepared by mixing of an aqueous solutions of Ag ions (0.5 mL, 0.1 mM) with aqueous solution of sodium citrate (12 mL, 0.1 mM). Then, aqueous solution of NaBH4 (0.5 mL, 0.1 M) was added into the above solution to form a stable colloidal solution of AgNPs. Imipenem (Exir, Iran) stock solution were prepared in 0.01 Mphosphate buffer and diluted as described by CLSI protocol. Bismuth ethandithiol (BisEDT) and Bismuth propanedithiol (BisPDT) were two biocides which was used in this study. For this reason, 4.58 mg of bismuth nitrate (BN) was dissolved in 1 mL of 1,2-propandiol. Either 100 µL of 1,2-ethanedithiol [EDT, (Merck, Germany)] or 100 µL of 1,2-propanedithiol [PDT, (Merck, Germany)] was dissolved in 900 µl of 1,2-
  • 3. Nanomed. J., 2(4): 291-298, Autumn 2015 293 propandiol. Then, 1000 µg/mL working stock solution of each biocide was prepared by adding 50 µl of BN, 5.8 µlofHCl(12.1 M)and4.2µlofEDT orPDT to940µl of 1,2-propandiol (20). MIC determination of silver nanoparticles on planktonic bacteria To determine the minimum inhibitory concentration of AgNPs on planktonic bacteria, serial dilutions of AgNPs were prepared by diluting 15 times of 100 ppm solution of AgNPs to reach to the half concentration.Suspension of cells in Muller Hinton broth was adjusted to 0.5 McFarland tubeand diluted 1:100 in Muller Hinton broth medium. Then, 100 µl of diluted AgNPs was added to each row of microtiter plate followed by addition of 100 µL of each isolates to the wells. Finally, microtiter plates were incubated at 37°C for 24 h (21).Assessment of synergetic effect of AgNPs in combination with imipenem on planktonic bacteria was determined by using checkerboard microdilution technique. Stock solutions of imipenemandAgNPsweredilutedand 50µLofimipenem dilutions (0.25-1024 µg/mL) were added to each row of microtiter plate.Afterward, 50 µLofAgNPs (0.003-100 ppm) were added to each column of microtiter plates. Then, 100 µL of each bacterium was added to each well. Plates were incubated at 37°C for 24 h (22).After that, the fractional inhibitory concentration (“FIC) for both antimicrobials was calculated according to European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (2000): “FIC d”0.5 is a sign of synergism, >0.5-1 sign of addition, >1 to 2 indifference and e”2 is a sign of antagonism. MIC determination of silver nanoparticles against biofilm For determination of minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of nanoparticles, 10 isolates out of 75 isolates of A. baumanniiwere chosen. The criterion for theselection was the ability of these strains to form strong biofilms as described in previously study(23). To determine MBIC of silver nanoparticles,serially diluted nanoparticles were prepared in TSB. Subsequently, microtiter plates were filled and incubated by AgNPs and suspension of cells, respectively as described for planktonic cells. Subsequently, plates were emptied and washed 5 times with phosphate buffer saline (PBS) and were left to dry. This step was followed by adding 200 µL of 0.2 % crystal violet. After 5 minutes; the plates was washed for 5 times and destained by using 33 % (v/v) glacial acetic acid. Next, 100 µL of each well contents were transferred to the wells of a new microtiter plate and the plates were read by an ELISA plate reader (24). The efficacy of biocides was calculated according to the following equation. Where B denotes the average of absorbance for blank wells, C denotes the average of absorbance for control wells and T denotes the average of absorbance for treated wells (25). To determine MBIC of AgNPs in combination with imipenem, Bismuth-ethandithiol(BisEDT) and Bismuth- propanedithiol (BisPDT) stock solutions of each antimicrobial were prepared in tryptic soy broth [TSB, (imipenem0.125-1024µg/mL,BisEDTandBisPDT0.09- 1000 µg/mL)] and synergetic effect of AgNPs as well fractional biofilminhibitoryconcentration (“FBIC) were determined. To determine minimum biofilm eradication concentration (MBEC), each microtiter plate was filled with given concentration of isolates and incubated for 24 h at 37°C to form biofilm. Next, plates were emptied and washed 5 times to remove non-biofilm forming cells (25). To determine of MBEC ofAgNPs,serially diluted concentrations of AgNPs were added to each well and incubated at 37 ° C for 20 minutes. Then plates were emptied and filledagain and repeated to get the final time of 1h.Afterward, plates were emptied and washed 5 times and followed by adding up neutralizer for 5 minutes (25). The sodium salt of XTT (2,3-Bis-(2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide)(Sigma) was dissolved in phosphate buffer saline to the concentration of 1 mg/mL. In addition, menadione (Sigma) was dissolved in acetone to reach to 1 mM concentration. The XTT/menadione
  • 4. Nanomed. J., 2(4): 291-298, Autumn 2015 294 Effect of AgNPs against Acinetobacter biofilms reagent must be prepared fresh prior to each assay and contained 12.5 parts XTT/1 part menadione. Then, 100 µL XTT-menadione were added to each well and incubated at 37°C in a shaker incubator at 150 r.p.m for 1h. Finally, absorbance at 505 nm were read by plate ELISA reader (26).The efficacy of biocide was calculated as described above. To determine of MBEC ofAgNPs in combination with imipenem, diluted antimicrobials were added to plates via checkerboard method and fractional biofilm eradication concentration (“FBEC) were then determined. Scanning electron microscopy was applied to determine effectiveness of antimicrobial agents to eradicate b i o f i l m . G l a s s s l i d e s w i t h d i m e n s i o n o f 1 × 1 c m 2 were placed in the wells of a 12-well sterile microtiter plate. The steps was followed by adding 3 mL of bacterial suspension and mediumin the first well, 1.5 mL bacterial suspension and 1.5 mL AgNPs in the second well and 1.5 mL bacterial suspension and 1.5 mLAgNPs and imipenem into the third well. The plate was incubated at 37°C for 24 h. After that, slides were washed by sterile distilled water and placed in Glutaraldehyde 4% for 2 h to fix. Then, to dehydrate all slides, they were put for 15 min in the serial concentrations of 40 %, 60 %, 80 %, 90 % and 100 % of absolute ethanol (Merck, Germany), respectively. Finally, biofilms on the surface of the glasses were lyophilized. Electron microscopic micrographs were taken using Phillips XL 30. Statistical analysis Results were analyzed with SPSS software using one way ANOVA. All experiments were performed in triplicate. P values of d” 0.05 were regarded as significant. RESULTS In this study, MIC of AgNPs against planktonic bacteria was determined. Only 22 isolates (29.3%) showed susceptibility to 100 ppm and remaining isolates were resistant to all AgNPs concentrations. Also, MBIC of AgNPs alone and in combination with imipenem, BisEDT and BisPDT was determined (Fig. 1). The concentrations of AgNPs, imipenem, BisEDT and BisPDT for all isolates has been shown in Table 1. As showed in scanning electron microscopy micrographs, AgNPs alone could reduce biofilm of A. baumannii isolates, but in combination with imipenem, biofilm producing isolates could not attach to the surface of glass and form the biofilm (Fig. 2). In the other hand, in comparison with AgNPs alone, AgNPs in combination with imipenem and biocides could inhibit biofilm formation of A. baumannii at lower concentrations. Moreover, results indicated synergistic activity of AgNPs and imipenem. However, antagonistic impact of AgNPs with BisEDT and BisPDT was clearly observed. MBEC of AgNPs alone and in combination with imipenem were also determined and presented in Fig. 3. Since generation time of Acinetobacter isolates was reported 20 minutes in previous(27), it seems after induction of cell division, AgNPs can inhibit this process resulting in cell death. As it was shown in the Table 1, synergistic impact between AgNPs and imipenem to eradicate biofilms was observed. Furthermore, statistical analysis showed significant differences between AgNPs alone and in combination with antimicrobials data with P values of d” 0.05. Finally, the combination of AgNPs and imipenem at lower concentrations could be inhibited biofilm formation of A.baumannii. Fig. 1. MBIC of AgNPs alone and in combination with imipenem, BisEDT and BisPDT was shown
  • 5. Nanomed. J., 2(4): 291-298, Autumn 2015 295 Table 1. Concentrations (µg/mL) of AgNPs, imipenem.BisEDT and BisPDT of MBIC were presented isolate 1 2 3 4 5 6 7 8 9 10 AgNPs AgNPs 0.8 6.5 1.6 6.5 6.5 6.5 6.5 0.8 0.8 6.5 AgNPs + IM1 AgNPs 0.0003 0.8 0.4 0.0015 0.0003 0.0031 0.0003 0.2 0.0003 0.0003 IM 0.25 32 32 8 0.125 32 1 16 16 16 AgNPs + BisEDT AgNPs 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.4 BisEDT 16 8 32 32 32 16 16 32 16 16 AgNPs + BisPDT AgNPs 0.2 0.4 0.2 0.2 0.2 0.2 0.2 0.4 0.2 0.2 BisPDT 16 16 32 16 16 16 16 16 16 32 1 IM = imipenem Fig. 2. Scanning electron micrographs of (a) and (b) positive control with different magnification, (c) and (d) treated the isolates with AgNPs alone with different magnification, (e) mixed AgNPs and imipenem treatment against the same isolate number 10
  • 6. Nanomed. J., 2(4): 291-298, Autumn 2015 296 S. Hendiani et al. Fig. 3. MBEC of AgNPs alone and in combination with imipenem DISSCUSION It was suggested that the positively charged AgNPs can attach to the lipopolysaccharides of Gram-negative bacteria and to be effective via metal reduction(29), However, Hong et al. reported that negativelycharged AgNPs can be electrostatically repulsed from the negatively charged (-20 mV) surfaces of bacteria (30). This feature could be attributed to the long time exposure (for example 24 hours) of nanoparticles in which resistance mechanisms like efflux systems could be deactivated the antimicrobial activity of AgNPs. In this study, synergistic effect of AgNPs in combination with imipenem(“FICd” 0.05) against allisolates was observed. According to results of MBIC, it could be suggested that the excretion of adhesive substances such as polysaccharides and proteins is crucial factor for the initial attachment of organisms as well to hold the bacteria together. It was reported that the AgNPs might be involved in preventing these adhesive substances and thereby resulting in prevention of biofilm formation(31). Since A. baumannii biofilm matrix comprises of extracellular DNA (eDNA),itseems that reaction between positively chargedAgNPs and negatively charged eDNA play a major role in the inhibitionof biofilm formation in isolates. BTs are the new classes of anti-biofilm agents that has been shown to have remarkable activity against a broad spectrum of biofilm producing organisms through inhibiting the slim expression in both Gram-positive and Gram-negative bacteria at non-toxic concentrations (32). In this study, it was assumed that the combination of AgNPs and BisEDT or BisPDT have a synergistic impact. Although both biocides are exopolysaccharide inhibitors but they have antagonistic activity when used together. The synergism between AgNPs and imipenem could be due to the damage of the matrix and consequently improved penetration of antibiotic. Furthermore, antibiotic molecules have many active sites such as hydroxyl and amine groups which can interact withAgNPs and act as a chelating agent. Moreover,imipenem lyses the cell wall of bacteria leading to increase in penetration of AgNPs into the cells. Thiols can chelate bismuth and other metals. It seems in the presence of AgNPs, thiols preferably bond to silverionsleading to easier penetration of Ag into the cells. Therefore, lower concentrations ofAgNPs when mixed with thiols, can inhibit biofilm formation. However, higher concentrations of thiol would be required when there is antagonism (“FBIC > 2) between AgNPs and thiols. CONCLUSION This study demonstrated a synergistic effect of imipenem andAgNPs against planktonic cells as well biofilms, making it a promising candidate for a new treatment of infections caused by MDR A. baumannii strains. Further investigation should be carried out to be sure of effectiveness of this nanoparticle in vivo experiments and assay probable cytotoxicity of this material. ACKNOWLEDGEMENTS The authors wish to thank Vice Chancellor of Alzahra University and Iran nanotechnology initiative council for the financial support and Burn and Army hospitals for providing the bacterial isolates. CONFLICTOFINTEREST No conflict of interest. REFERENCES 1. Panacek A, Kvítek, L., Prucek, R., Kolar, M., Vecerova, R., Pizúrova, N., Sharma, V, K., Nevecna, T., Zboril, R. Silver colloid nanoparticles: synthesis, characterization, and their
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  • 8. Nanomed. J., 2(4): 291-298, Autumn 2015 298 S. Hendiani et al. 30. Hong S, H., Jeong, J., Shim, S., Kang, H., Kwon, S., Ahn, K, H., Yoon, J. Effect of electric currents on bacterial detachment and inactivation. Biotechnol Bioeng. 2008; 100(2): 379-89. 31. Pratik R, Chaudhari, Shalaka, A., Masurkar, Vrishali, B., Shidore, Suresh, Kamble, P. Effect of Biosynthesized Silver Nanoparticles on Staphylococcus aureus Biofilm Quenching and Prevention of Biofilm Formation. Nano-Micro Lett 2012; 4(1): 34-9. 32. Gunawardana J. Bismuth-Ethandithiol: a potential drug to threat Biofilm infections of medical devices produced by Staphylococcus aureus and Proteus mirabilis. Florida, USA: Boca Raton; 2010. How to cite this article: Hendiani S, Ahya Abdi A, Mohammadi P, Kharrazi Sh. Synthesis of silver nanoparticles and its Synergistic effects in combination with imipenem and two biocides against biofilm producing Acinetobacterbaumannii. Nanomed. J., 2015; 2(4): 291-298.