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                    International Journal of Nanomedicine

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Effects of silver nanoparticles in combination with
antibiotics on the resistant bacteria Acinetobacter
baumannii
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
12 August 2016
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Guoqing Wan 1,2
Lingao Ruan 2,3
Yu Yin 2,3
Tian Yang 2,3
Mei Ge 2
Xiaodong Cheng 1,4
School of Life Science and
Technology, China Pharmaceutical
University, Nanjing, 2Shanghai Laiyi
Center for Biopharmaceutical R&D,
3
School of Pharmacy, Shanghai Jiao
Tong University, Shanghai, People’s
Republic of China; 4Department of
Integrative Biology & Pharmacology,
The University of Texas Health
Science Center, Houston, TX, USA
1

Correspondence: Mei Ge
Shanghai Laiyi Center for
Biopharmaceutical R&D, No 800,
Dongchuan Road, Shanghai 200240,
People’s Republic of China
Tel/fax +086 21 3420 4838
Email gemei@yeah.net
Xiaodong Cheng
Department of Integrative Biology &
Pharmacology, the University of Texas
Health Science Center, Houston, USA
Tel/fax +00171 3500 7487
Email xiaodong.cheng@uth.tmc.edu

Abstract: Acinetobacter baumannii resistance to carbapenem antibiotics is a serious clinical
challenge. As a newly developed technology, silver nanoparticles (AgNPs) show some excellent
characteristics compared to older treatments, and are a candidate for combating A. baumannii
infection. However, its mechanism of action remains unclear. In this study, we combined AgNPs
with antibiotics to treat carbapenem-resistant A. baumannii (aba1604). Our results showed that
single AgNPs completely inhibited A. baumannii growth at 2.5 μg/mL. AgNP treatment also
showed synergistic effects with the antibiotics polymixin B and rifampicin, and an additive effect
with tigecyline. In vivo, we found that AgNPs–antibiotic combinations led to better survival
ratios in A. baumannii-infected mouse peritonitis models than that by single drug treatment.
Finally, we employed different antisense RNA-targeted Escherichia coli strains to elucidate the
synergistic mechanism involved in bacterial responses to AgNPs and antibiotics.
Keywords: Acinetobacter baumannii, AgNPs, synergistic, antibiotic combination, anti­
sense RNA

Introduction
Drug-resistant Acinetobacter baumannii is an infectious pathogen that currently presents
serious clinical challenges. A. baumannii is particularly associated with hospital-acquired
infections such as pneumonia, bloodstream, abdominal, central nervous system, urinary
tract, and skin and soft tissue infections.1 A. baumannii can develop resistance against
antibiotics through several mechanisms;2 in particular, this bacterium is often resistant
to the carbapenems.3 Increasing numbers of carbapenem-resistant A. baumannii isolates
have been reported worldwide.4 The majority of such bacteria are extensively drug
resistant, which may include resistance to carbapenems and all other antibiotics except
polymyxins and tigecycline.5 Polymyxin B is effective against drug-resistant A. baumannii, but systemic application carries risk of toxicity, primarily kidney toxicity and
neurotoxicity.6,7 A. baumannii infection is common in patients with severe infections,
and is often accompanied by other bacterial and/or fungal infections.8 Patients infected
with resistant A. baumannii have high mortality.9 Therefore, there is an urgent need to
find suitable therapeutic drugs for the treatment of resistant A. baumannii infections.
The ineffectiveness of synthetic antibiotics against drug-resistant bacteria has led
to the reemergence of interest in silver, which has an ancient history as an antibacterial
agent.10–12 The antibacterial activity of silver nanoparticles (AgNPs) had been reported
against multiple species of bacteria; for example, Escherichia coli ATCC 8739,13 Staphylococcus aureus ATCC1431,14 Escherichia fergusonii, and Klebsiella aerogenes ATCC
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http://dx.doi.org/10.2147/IJN.S104166

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Wan et al

1950,15 among others. Synthesized AgNPs with capping
agents, such as citrate, sodium dodecyl sulfate, and polyvinylpyrrolidone show increased antibacterial activity against S.
aureus and E. coli.16 Huang et al reported antimicrobial activity against A. baumannii with the synergistic combination of
chitosan acetate and AgNPs.17 Jain et al studied the interaction
of AgNPs with commonly used antibiotics in Pseudomonas
aeruginosa,15 while Morones-Ramirez et al demonstrated
that Ag+ treatment sensitized Gram-negative bacteria to the
Gram-positive-specific antibiotic vancomycin, both in vitro
and in vivo.18 However, the synergistic antimicrobial activity
of antibiotics combined with citrate-capped AgNPs has yet
to be studied.
In the present study, we investigated the synergistic
combinatorial effects of antibiotics with AgNPs against
drug-resistant A. baumannii obtained from clinical patients
both in vivo and in vitro. We also investigated the possible
mechanisms of this synergistic effect.

Materials and methods
Materials
Trisodium citrate, silver nitrate (AgNO 3), and sodium
borohydride (NaBH 4) were used for the synthesis of
AgNPs. Rifampicin, tigecyline, polymyxin B (PMB),
mucin, dimethyl sulfoxide, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), isopropylβ- d -thiogalactoside, penicillin–streptomycin, and
trypsin–ethylenediaminetetraacetic acid were purchased
from Sigma Aldrich (St Louis, MO, USA). A549 cells and
HL-7702 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, People’s
Republic of China). Roswell Park Memorial Institute-1640
medium was purchased from Thermo Fisher Scientific
(Waltham, MA, USA) and fetal bovine serum was purchased
from HyClone (Logan, UT, USA). No ethical committee
approval was required for this set of experiments because the
experiments were performed on commercially available cell
lines and were considered exempt from full review by the
ethics Committee at Shanghai Jiao Tong University.
All animal procedures were approved by the Institutional
Animal Care and Use Committee at Shanghai Jiao Tong
University. All the animal studies were performed according
to the Guiding Principles for the Care and Use of Laboratory Animals according to the Regulations of the People’s
Republic of China for Administration of Laboratory Animals.
All animal procedures were approved by the Animal Ethics
Committee of Shanghai Jiao Tong University. C57BL/6
mice were purchased from Slac Laboratory Animal Co., Ltd.
(Shanghai, People’s Republic of China).
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Synthesis and characterization of AgNPs
In a three-necked round-bottomed flask, 20 mL trisodium
citrate (1%) and 75 mL ultrapure water were mixed for
15 minutes at 70°C. To the solution, 1.5 mL of silver nitrate
solution (1%) was added; NaBH4 (1%) was then added,
followed by rapid mixing. This mixed solution was heated
for 60 minutes, cooled to room temperature, and water was
added to a volume of 100 mL.
To characterize the morphology of the synthesized
AgNPs, transmission electron microscopy (TEM) analysis
was performed using a Tecnai G2 Spirit 120 kV TEM
instrument (0.23 nm resolution) (FEI Company, Hillsboro,
OR, USA).
AgNPs were further characterized by scanning the absorbance spectra in 300–500 nm range of wavelength with a
multifunction full wavelength microplate analyzer (BioTek
Co., Winooski, VT, USA).
A Malvern Zetasizer Nano-ZS instrument (Malvern,
Louis, USA) was used to characterize the zeta potential of
the nanoparticles in the solution. Data were obtained and
analyzed using Zetasizer software (Malvern, Louis, USA).

Determination of minimum inhibitory
concentration and fractional inhibitory
concentration
The bacterial strain A. baumannii (aba 1604; Fudan University
Huashan Hospital, Shanghai, People’s Republic of China)
was used as a model test strain to determine the antibacterial
activity of AgNPs. Various concentrations of AgNPs were
incubated with 4×105 bacteria in Luria Bertani (LB) broth
medium in 96-well round-bottomed plates. Bacteria were
harvested at the indicated time points and the optical density
of the samples was assayed at 600 nm. All samples were
plated in triplicate, and values were averaged from three independent trials. The resistant A. baumannii strain (aba1604)
was made from clinical patients and following institutional
ethical guidelines that were reviewed and approved by the
ethics committee at the Huashan hospital clinical ethics
committee, Fudan University. Consent from clinical patients
was not deemed necessary by the Shanghai Laiya centre for
Biopharmaceutical R & D who obtained these strains for
antibacterial drug research and collected these samples, as
the sample collection was part of normal patient care.
To evaluate the antibacterial activity of AgNPs in combination with antibiotics, a two-dimensional microdilution
assay was used.19 Assays were carried out in LB broth growth
medium. Minimum inhibitory concentration (MIC) for each
of the antibiotics was first estimated, and the fractional
inhibitory concentration (FIC) of a combination of antibiotics
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Effects of AgNPs with antibiotics on Acinetobacter baumannii

and AgNPs was subsequently determined by the checkerboard
microtitration method in a 96-well microtiter plate. Antibiotics and AgNPs were diluted to the following concentrations
(2MIC, 1MIC, 1/2MIC, 1/4MIC, 1/8MIC, 1/16MIC, and
1/32MIC) in the two-dimensional microdilution assay.
The plates were incubated at 37°C for 18 hours, and
results were assayed by measuring the optical density (OD)600.
The combined antibiotic effect of agents A and B (where A is
either AgNO3 or AgNPs, and B is one of three antibiotic
agents) was calculated as follows:
The FIC index:
MIC (A in combination with B)
=
MIC (A alone)
MIC (B in combination with A)
+
MIC (B alone)

access to food and water. Ten mice per group were given intraperitoneal injections of 100 μL total volume. Mice were treated
as follows: no treatment, and 10, 20, 40, and 80 mg/kg AgNPs
and AgNO3. Injected animals were observed for 3 days.

Determination of minimum lethal dose of
A. baumannii for peritonitis mouse model
Serial dilutions of A. baumannii ranging from 1×107 to 1×1011
CFU, in 500 μL sterile saline supplemented with 8% mucin,
were injected into the peritoneal cavity of mice.18 Animals were
observed for 2 days for determination of the survival rate.

Survival assays

FIC index values above 4.0 indicate antagonistic effects,
values between 0.5 and 4.0 indicate additive effects, and values
lower than 0.5 indicate synergistic effects.20

Mice received intraperitoneal injections of the minimum
lethal dose (MLD) of A. baumannii, with a total volume
of 500 μL with 8% mucin. After 1 hour, ten mice in each
group received a 100 μL intraperitoneal injection of either
vehicle phosphate buffer saline (PBS) or one of the different
antibacterial treatments. Mice were observed for 2 days to
evaluate the survival rate.

Cytotoxicity assay

Bacterial colonization assays



(1)

To determine the cytotoxic activity of the AgNPs on mammalian cells, A549 cells and HL-7702 cells (1×104 cells/mL)
were grown in Roswell Park Memorial Institute-1640 medium
containing 5% fetal bovine serum in a 96-well plate at 37°C
in an atmosphere of 5% CO2 for 24 hours. Cells were treated
with AgNPs, AgNO3, or control solutions at concentrations
ranging from 0.625 to 10 μg/mL for another 24 hours. To
determine the viability, MTT (at a concentration of 0.1 mg/mL)
was added to the wells and incubated for 4 hours at 37°C and
5% CO2 to allow cell growth.21 In metabolically active cells,
MTT was reduced to an insoluble, dark purple formazan. The
purple formazan was then dissolved in dimethyl sulfoxide. The
absorbance was measured at 570 nm using a multifunction full
wavelength microplate analyzer and readings were compared
from untreated cells. The OD values were used to sort out the
percentage of viable cells by using the following formula:
Percentage OD value of experimental sample
=
×100 (2)
OD value of experimental
of viability
control (untreated)


Assay for antimicrobial activity in vivo

Minimum lethal dose of AgNO3 or AgNPs in mice

Six-week-old male C57BL/6 mice (body weight ~20 g)
were used for all animal experiments. Mice were housed in a
temperature- and humidity-controlled environment, and had free
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Surviving mice were euthanized and dissected, and their kidneys and lungs were collected. These organs were ground under
aseptic conditions, and the homogenates were dissolved in
sterilized saline water. These organ homogenates were then
cultivated on LB plates at 37°C for 24 hours.

Cytokine profiling
Cytokine concentrations in the mouse plasma were measured
at the indicated time after infection by standard enzymelinked immunosorbent assay kits following the manufacturer’s instructions (Elabscience Biotechnology Co., Ltd,
Wuhan, People’s Republic of China).

Antisense RNA models for detecting
the synergistic mechanism of AgNPs
and antibiotic combinations
To investigate the pathways involved in the bacterial response
to AgNPs, we conducted a series of experiments in which the
impacts of AgNPs and AgNO3 on different E. coli antisense
RNA-induced gene-silencing strains were examined.22–24 The
various gene-silenced strains were arrayed in microwell plates,
and then screened to determine how their sensitivity to each of
the different Ag formulations compared to the parent strain.
The gene-silenced E. coli strains were treated with isopropylβ-d-thiogalactoside at appropriate concentrations (Table 1),
and portions of the culture were transferred into 96-well plates.
Sublethal concentrations of AgNPs, AgNO3, rifampicin,
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Table 1 The optimal concentration of IPTG for antisense RNA strains
Genes

Target genes function

IPTG (μmol/L)

ligA
dnaB
rpsR
rpsA
rpsL
rplC
rplT
rplS
rpmA
murA
murB
murG
murE
leuS
tufA
rpoD
fabI
kdsA
kdsB
lpxC
lepB
mutL
menD

DNA biosynthesis
Replicative DNA helicase, DNA biosynthesis
Small subunit ribosomal protein S18, ribosome
Small subunit ribosomal protein S1, ribosome
Large subunit ribosomal protein L7/L12, ribosome
Large subunit ribosomal protein L3, ribosome
Large subunit ribosomal protein L20, ribosome
Large subunit ribosomal protein L19, ribosome
Large subunit ribosomal protein L27, ribosome
UDP-N-acetylglucosamine 1-carboxyvinyltransferase, peptidoglycan biosynthesis
UDP-N-acetylenolpyruvoylglucosamine reductase, peptidoglycan biosynthesis
Peptidoglycan biosynthesis
UDP-N-acetylmuramoylalanyl-d-glutamate–2,6-diaminopimelate ligase, peptidoglycan biosynthesis
Leucyl-tRNA synthetase, aminoacyl-tRNA biosynthesis
Elongation factor Tu, protein biosynthesis
Principal σ factor , RNA biosynthesis
Enoyl-[acyl-carrier protein] reductase I, fatty acid biosynthesis
Lipopolysaccharides biosynthesis
Lipopolysaccharides biosynthesis
Lipopolysaccharides biosynthesis
Signal peptidase, protein export
DNA mismatch repair protein MutL, mismatch repair
Ubiquinone and other terpenoid-quinone biosynthesis

800
20
10
40
10
20
40
40
60
20
40
40
40
40
40
40
40
1,600
1,600
1,600
40
40
40

Abbreviations: IPTG, isopropyl-β-d-thiogalactoside; tRNA, transfer RNA; UDP, uridine diphosphate.

tigecyline, and PMB were added to the gene-silenced bacteria
in 96-well plates. Finally, the plates were incubated at 37°C
for 16 hours and shaken at 80 rpm. Absorbance was measured
at 600 nm using a multifunction full wavelength microplate
analyzer, and OD values were used to calculate the inhibition
ratio (I) by using the following formula:
I (%)
OD value of control (untreated) − OD value of sample
× 100
=
OD value of control (untreated)



(3)

∆I (%) = I antisense RNA-induced gene-silencing strains − I E .coli DH5α /pHN678 (4)


Statistical analysis
Each assay was repeated three times. Data are presented
as mean ± standard deviation, unless otherwise noted.
Comparisons between multiple groups were made using
one-way analysis of variance (ANOVA) and all analyses
were performed using SPSS 21.0 statistical software (21.0;
IBM Corporation, Armonk, NY, USA). The threshold for
statistical significance was set at P,0.05.

Results
Characterization of AgNPs
Based on the image in Figure 1A, AgNPs stabilized by
citrate had good dispersion. The particle size distribution was
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shown by counting AgNPs particle numbers based on TEM
images and the histogram of particle size distribution was
in Figure 1B. AgNPs were 5–12 nm in diameter, with an
average size of 8.4 nm. The nanoparticles were found to be
stable for over 6 months, even at 37°C. The zeta potential
of the synthesized AgNPs is summarized in Figure 1C.
The ultraviolet–visible (UV–vis) spectra of the solution
samples are reported in Figure 1D. A single strong peak was
observed at 392 nm, which indicates the synthesis of spherical nanoparticles. In practice, dispersion was stable if the
zeta potential was higher than 30 mV or less than −30 mV.
Supporting the earlier statement, we observed that AgNPs
dispersed in water were highly stable with a zeta potential
value of −44.5 mV.

Antibacterial activities of AgNP
combination treatments
Combination antibiotic therapy is a strategy often employed
in the treatment of multiple drug resistance (MDR) A. baumannii. Because PMB, rifampicin, and tigecycline are all
commonly used against MDR A. baumannii in combination
with other antibiotics,25–27 we selected these three antibiotics to evaluate potential combinatorial effects with AgNPs.
It was seen that AgNPs displayed potent antimicrobial
activity against A. baumannii, with an MIC of approximately 2.5 μg/mL, similar to that of AgNO3. Typical drug
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Effects of AgNPs with antibiotics on Acinetobacter baumannii

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Figure 1 Appearance and physicochemical characteristics of AgNPs.
Notes: (A) TEM of AgNPs. (B) Size distribution of AgNPs based on TEM images. (C) Zeta potential analysis of AgNPs. (D) UV–visible absorption spectroscopy showed the
maximum absorbance at 392 nm for AgNPs.
Abbreviations: AgNPs, silver nanoparticles; OD, optical density; TEM, transmission electron microscopy; UV, ultraviolet.

MICs, summarized in Table 2, were 0.25 μg/mL for PMB,
3.12 μg/mL for rifampicin, and 3.12 μg/mL for tigecycline.
The FIC of AgNPs and the various antibiotic combinations were investigated and are summarized in Table 2.
These experiments showed that PMB and rifampicin acted
synergistically (P,0.5) with AgNPs and AgNO3, while
tigecycline did not show synergy (P.0.5) with either AgNPs
or AgNO3.
Table 2 FIC index of combinations among silver and antibiotics
against Acinetobacter baumannii
Compounds

FIC of
AgNPs

FIC of
AgNO3

MIC of antibiotics
(µg/mL)

Polymyxin B
Rifampicin
Tigecycline
AgNPs
AgNO3

0.19
0.38
0.75
–
–

0.19
0.38
0.75
–
–

0.25
3.12
3.12
2.5
2.5

Note: Data are presented as mean ± SD, unless otherwise specified.
Abbreviations: FIC, fractional inhibitory concentration; MIC, minimum inhibitory
concentration; AgNPs, silver nanoparticles; SD, standard deviation.

International Journal of Nanomedicine 2016:11

Cytotoxicity of AgNPs in vitro
Many investigations have reported on the inhibitory effects
of AgNPs on cells. For example, Beer et al found that AgNPs
inhibited the proliferation of A549 cells in a dose-dependent
manner,28 whereas Foldbjerg et al reported that AgNPs
induced increase in reactive oxygen species (ROS) level in
A549 cells.29 Here, we used the method described by Foldbjerg et al to evaluate the cytotoxicity of AgNPs.29
As shown in Figure 2, high concentrations of AgNO3
significantly affected cell growth. By comparison, exposure
to AgNPs at a higher concentration of 10 μg/mL did not
exhibit significant cytotoxicity in A549 and HL-7702 cells.
These results demonstrate that the cytotoxicity of AgNPs is
lower than that of AgNO3.

Effects of AgNPs on antimicrobial activity
in vivo
The acute toxicity of AgNPs and AgNO3 were measured
in vivo to establish the median lethal dose (LD50) (Figure 3).
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Figure 2 Relative survival of A549 and HL-7720 cells exposed to AgNPs.
Notes: Relative survival of cells as affected by different doses AgNPs or AgNO3. (A) MTT assay results confirmed the in vitro cytotoxicity of AgNPs and AgNO3 against A549
cells. (B) Effects of AgNPs or AgNO3 on HL-7720 cell growth. Results are shown as the mean ± SD of three independent experiments. *P,0.05, **P,0.01 and ***P,0.001
vs control.
Abbreviations: AgNPs, silver nanoparticles; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation.

The LD50 of AgNPs was determined to be between 20
and 40 mg/kg, and the LD50 of AgNO3 was also found
to be between 20 and 40 mg/kg. These LD50 values for
AgNO3 are similar to those reported in an earlier toxicity
study.18
Infection was established in mice through intraperitoneal
delivery of 5×109 A. baumannii cells suspended in an aqueous
solution containing 8% mucin. Within 24 hours after the time
of injection, all infected mice had died. Thus, A. baumannii
was used at this same concentration of 5×109 cells for all
subsequent experiments.
The PMB and AgNPs combination and the PMB and
AgNO3 combination showed the same synergistic antibiotic

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effects that we had earlier observed in vivo (Figure 4). We
observed the mice living condition for 1 week, and calculated 2 days survival rate. A mixture of AgNO3 and PMB
(3 mg/kg and 10 μg/kg, respectively) resulted in a survival
rate of 40%. When either of these two compounds was given
alone as a single-dose treatment, survival rates were 0%.
By comparison, even a low-dosage mixture of AgNPs and
PMB (2 mg/kg and 10 μg/kg, respectively) resulted in a high
survival rate of 60%.
Two days after administration in mice, we dissected a
portion of mice for bacterial colonization assay. Representative images of bacterial growth are presented in Figure 5,
which show that AgNO3 and AgNPs enhanced the action of

























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Figure 3 Toxicity of AgNPs and AgNO3 in mice.
Notes: (A) Survival of mice given the following treatments: control, 10, 20, 40, and 80 mg/kg AgNO3. (B) Survival of mice given the following treatments: control, 10, 20,
40, or 80 mg/kg AgNPs. Survival assays were performed with ten mice per group.
Abbreviations: AgNPs, silver nanoparticles; h, hours.

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Effects of AgNPs with antibiotics on Acinetobacter baumannii

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Figure 4 Survival of mice given AgNPs or AgNO3 with PMB in a peritonitis infection model.
Notes: (A) PMB treatment concentration in peritonitis infection model. (B) AgNO3 and PMB treatment concentration in peritonitis infection model. (C) AgNPs and PMB
treatment concentration peritonitis infection model.
Abbreviations: AgNPs, silver nanoparticles; PMB, polymyxin B; h, hours.

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Figure 5 Bacterial burdens in Acinetobacter baumannii-infected mice after treatment with PMB combined with AgNO3 or AgNPs.
Notes: Bacterial burdens in Acinetobacter baumannii-infected mice after treatment with PMB (250 μg/kg) (a); the infected mice after treatment with PMB combined with
AgNO3 (3 mg/kg, 18 μM) (b1: 250 μg/kg; b2: 50 μg/kg; b3: 10 μg/kg), and with AgNPs (2 mg/kg, 18 μM) (c1–c3). (A) kidney; (B) lung; (C) blood; (D) ascitic fluid.
Abbreviations: AgNPs, silver nanoparticles; PMB, polymyxin B.

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Figure 6 AgNO3 or AgNPs combined with antibiotics-modulated Acinetobacter baumannii-induced inflammatory reaction.
Notes: ELISA was used to measure IL-6 (A) and TNF-α (B) in the mouse plasma 18 hours post infection. Results are shown as the mean ± SD of three independent
experiments. ***P,0.001 vs control; #P,0.05 and ##P,0.01 vs infection model; $P,0.05 vs PMB only group. We obtained P-values by a Students’ t-test.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL, interleukin; PMB, polymyxin B; TNF-α, tumor necrosis factor alpha; SD, standard deviation.

PMB against A. baumannii in peritonitis infection model.
Figure 5A and B shows that there are plenty of bacteria
present in both the kidney and lungs when animals are
treated with PMB alone at a dose of 250 μg/kg. With the
addition of AgNO3 (3 mg/kg, 18 μM) or AgNPs (2 mg/kg,
18 μM), no bacteria were detected in the kidneys or lungs.
When we reduced the PMB dose to 50 μg/kg, we found that
the kidney and lung tissues contained only a small amount
of bacteria. Further reduction of the PMB dose to 10 μg/kg
was less effective, leaving a substantial bacterial burden in
the kidney and lungs. Moreover, we also tested the blood
and ascites of infected mice and found that they did not
contain bacteria under the combined administration of PMB
(50 μg/kg) with either AgNO3 (3 mg/kg, 18 μM) or AgNPs
(2 mg/kg, 18 μM) (Figure 5C and D). These results indicate
not only that AgNO3 can enhance the antibacterial activity
of antibiotics but also that AgNPs possess antibacterial
capability in vivo.
To analyze whether AgNO3 or AgNPs combined with
antibiotics regulate inflammation during A. baumannii
infection, we assessed proinflammatory cytokines in the
mouse plasma using enzyme-linked immunosorbent assay.
The levels of tumor necrosis factor alpha (TNF-α) and
interleukin (IL)-6 decreased significantly in the mouse
plasma of treated mice compared to those of model mice
at 18 hours post infection (Figure 6). AgNO3 and AgNPs
could enhance the action of PMB against A. baumannii
in vivo.

3796

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Figure 7 Heat map analysis of sensitivity to AgNPs and AgNO3 in gene-silenced
Escherichia coli strains relative to the control E. coli DH5α/pHN678 strain.
Notes: Each unit represents the difference in inhibition rate between a single
antisense RNA-induced gene silencing and the control E. coli DH5α/pHN678 strain.
Red units represent the most sensitive strains for compounds, and blue represents
strains that grow similarly to the control E. coli DH5α/pHN678 strain.
Abbreviations: AgNPs, silver nanoparticles; TIGE, tigecycline; RIF, rifampicin;
PMB, polymyxin B.

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Mechanisms for enhanced antimicrobial
activity of AgNPs in combination with
antibiotics
To better understand the mechanisms supporting the synergistic effects of AgNPs combined with antibiotics, we
used antisense RNA-induced gene silencing to silence the
expression of a number of genes in E. coli (Figure 7). The
difference in inhibition rate between a set of gene-silenced
E. coli strains and the control E. coli DH5α/pHN678 strain
is represented by color in our heat map analysis; dark red
represents the most sensitive strains, whereas dark blue represents the least sensitive strains, relative to the parent strain.
We found that silencing of rpoD, kdsA, kdsB, lpxC, and mutL
resulted in sensitivity to both AgNO3 and AgNPs. Silencing
of rpsR, rpsL, rpsA, murB, murA, leuS, and dnaB led only
to sensitivity to AgNO3, which indicates that AgNPs act at a
higher selectivity than AgNO3. In addition, the rpoD-silenced
strain was sensitive to rifampicin, while the silencing of kdsA,
kdsB, and lpxC increased sensitivity to PMB.

Discussion
A. baumannii has been proved to be resistant to many kinds of
antibiotics, which is suitable for genetic exchange. Fournier
et al reported an 86 kb genomic region naming AbaR1
resistance island in AYE which had 45 resistance genes
in the MDR isolate.30 The key resistance genes were those
coding for AmpC, VEB-1, and OXA-10 beta-lactamases,
tetracycline efflux pumps, and various aminoglycosidemodifying enzymes. Moreover, Fournier et al found that
17 out of the 22 clinical A. baumannii isolates showed an
original ATPase ORF. These 17 isolates contained eleven
isolates that are resistant to several antibiotic families, including β-lactams, and six other isolates susceptible to β-lactams.
AgNPs could damage the membrane potential, prevent ATP
production, increase the level of ROS, and damage the
membrane lipids as well as DNA, which demonstrated that
AgNPs have broad-range antibacterial properties, including
A. baumannii.31
It has been reported that citrate-capped AgNPs are less
toxic to mammalian cells and show increased antimicrobial
activity against S. aureus and P. aeruginosa.32 Our results
support these findings, and further build from them to explore
the toxicity of AgNPs to A549 and HL-7702 cells, and to
describe the effects of AgNPs on drug-resistant A. baumannii.
Our study demonstrates that the cytotoxicity of AgNPs is
lower than that of AgNO3.
The combination of AgNPs and either PMB or rifampicin
showed strong synergistic antimicrobial effects. Moreover,

International Journal of Nanomedicine 2016:11

Effects of AgNPs with antibiotics on Acinetobacter baumannii

the pairing of AgNPs and PMB showed an enhanced effect
against A. baumannii in vivo, which suggests the possibility
of a clinical application for this combination therapy.33
IL-6 and TNF-α are cytokines that have been shown
to play an important role in the host immune response
against intracellular pathogens in murine models. Smani
et al demonstrated that A. baumannii induced the release
of TNF-α and IL-6 and increased the Ca2+ influx.34 The
levels of TNF-α and IL-6 decreased significantly in the
mouse plasma of treated mice compared to those of model
mice, which further proved that inhibiting proinflammatory
signals could be protective during A. baumannii infections.
Sarkar et al reported that AgNPs could modulate human
macrophage responses to Mycobacterium tuberculosis.35 A.
baumannii is also highly correlated with the host immune
status.35 We have verified the role of AgNPs on A. baumannii. Hence, we hypothesized that AgNPs could modulate
human macrophage responses to A. baumannii, as the mode
of M. tuberculosis.
E. coli is one of the most representative model organisms
in experimental biology and medical study; there are a large
number of experimental studies on the drug mechanisms and
targets using E. coli.22–24 On the one hand, A. baumannii is
a common clinical pathogen, which has received increased
attention.36 A. baumannii and E. coli are Gram-negative
bacteria, and they have similar cell structure.37 So we probe
the mechanism of synergistic effect of AgNPs and antibiotics combination against A. baumannii using E. coli as a
model system.
The probable role of PMB in such drug synergy is its
rapid permeabilization of the outer cell membrane, allowing
enhanced penetration by AgNPs. Polymyxin B can displace
Mg2+ or Ca2+, and also binds to the Lipid A component of
lipopolysaccharide (LPS), resulting in changes to the outer
membranes of bacteria. Our gene-silencing experiments
also suggested that LpxC plays a role in sensitivity toward
AgNPs and AgNO3. Lin et al found that an LpxC inhibitor
blocked the ability of bacteria to activate the sepsis cascade, enhanced opsonophagocytic killing of A. baumannii,
and protected mice from lethal infection.38 Moreover, the
potential contributions of PMB and AgNPs both involve
Lipid A of LPS, a convergence which supports their synergistic effects.
KDO 2-keto-3-deoxyoctanoic acid (KDO) plays an
essential role in LPS biosynthesis, and may serve a universal
role in group 2 capsule biosynthesis by linking the polysaccharide region to the lipid domain.39 The kdsA gene encodes
the protein KDO 8-phosphate synthetase, which catalyzes

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Figure 8 Proposed mechanisms of the combination of AgNPs/Ag+ with antibiotics against G+ negative bacterium.
Notes: AgNPs/Ag+ can enhance PMB-induced damage of the membrane lipids. AgNPs/Ag+ and RIF also may bind intracellular proteins and RNA polymerase, upon entering
the cytosol.
Abbreviations: AgNPs, silver nanoparticles; PMB, polymyxin B; RIF, rifampicin; LPS, lipopolysaccharide; CMP-KDO, cytosine monophosphate-2-keto-3-deoxyoctanoic
acid.

the first step of the KDO synthetic reaction.40 The kdsB gene
encodes CMP-KDO synthetase, which is essential to the LPS
biosynthesis pathway.41 Our gene-silencing results suggested
that kdsA and kdsB are involved in bacterial sensitivity toward
AgNPs and AgNO3, as well as PMB.
Similarly, we observed that silencing of RpoD, which is
involved in promoter localization and plays a crucial role in
transcription initiation,42 increased the sensitivity of E. coli
toward AgNPs and AgNO3. Most of the factors investigated
belong to the σ70 family, and all bacteria express one or more
σ70 factors. The σ70 factor sequence is highly conserved
and plays an important role in bacterial growth. Rifampicin
has a molecular mechanism of action that involves inhibition of DNA-dependent RNA polymerase.43 In E. coli, this
enzyme is a complex oligomer comprising four subunits: α,
β, β′, and σ, encoded, respectively, by rpoA, rpoB, rpoC,
and rpoD, and their disruption interferes in the transcription
process.43 The potential mechanisms of both rifampicin and
AgNPs involve effects on DNA-dependent RNA polymerase,
which is the evidence that supports their synergistic effects
(Figure 8).
Silver and silver-containing compounds have recently
drawn increasing interest as antimicrobial agents for treating
bacterial infections. AgNPs showed synergy of inhibiting
P. aeruginosa biofilms when combined with sub-MIC levels

3798

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of aztreonam.44 Combination of AgNPs with ceftazidime
also showed a synergy to inhibit P. aeruginosa.45 AgNPs
prepared as described by Tiwari et al exhibited tremendous
antibacterial activity against a carbapenem-resistant strain of
A. baumannii.3 This required an efficient treatment regimen,
and the combination of rifampin with imipenem had been
evaluated in clinical infections caused by a highly imipenemresistant A. baumannii strain.46 Yoon et al showed that the
combination of PMB plus imipenem was as effective as PMB
plus rifampin against a carbapenem-resistant strain of A. baumannii.26 Therefore, drug treatment with newer antimicrobials or antimicrobial combinations has become increasingly
important to eradicate these infections. According to our
study, the combination of AgNPs with antibiotics could be
an effective solution to the problem of carbapenem-resistant
strains of A. baumannii, potentially at lower and less-toxic
doses than what is now typically used clinically.
Investigators have previously suggested that combination drug therapy could be an effective tool to prevent the
emergence of bacterial resistance, especially for patients
infected with Gram-negative bacteria that have developed
resistance to a single therapy. 47 Our study showed the
synergistic effects of combining AgNPs and PMB or AgNPs
and rifampicin against drug-resistant A. baumannii isolated
from clinical patients. Considering the lower toxicity of

International Journal of Nanomedicine 2016:11

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AgNPs compared to other treatment options, these drug
combinations have potential as useful tools for the clinic.

Acknowledgments
The authors are very grateful to Professor Zhu Demei
from Fudan University Huashan Hospital, who provided
the resistant A. baumannii strain (aba1604) from clinical
patients.
This work was funded by The National Major Scientific
and Technological Special Project for “Significant New
Drugs Development” (2012ZX09301002-003-007).

Disclosure
The authors report no conflicts of interest in this work.

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