TagedEnAmerican Journal of Infection Control 51 (2023) 871−878

Contents lists available at ScienceDirect

TagedFiur TagedEn American Journal of Infection Control TagedFiur TagedEn

journal homepage: www.ajicjournal.org

Major Article

TagedH1Antibacterial activity of silver nanoparticles functionalized with amikacin

applied against multidrug-resistant acinetobacter baumanniiTagedEn
agedPLT arissa de O. Camargo MS a, Inglid Fontoura MS a, Thaís S. Veriato MS a, Leandro Raniero PhD b,
Maiara L. Castilho PhD a,*TagedEn
a
TagedP Bionanotechnology Laboratory, Research and Development Institute, University of Paraiba Valley, Sa ~o Jose dos Campos/SP, Brazil
b ~o Jose dos Campos/SP, Brazil
Nanosensors Laboratory, Research and Development Institute, University of Paraiba Valley, Sa
TagedEn

TagedEnTagedPKey Words: TAGEDPA B S T R A C T

Silver nanoparticles
Multidrug-resistant Background: Multidrug-resistant bacteria are one of the world’s biggest health problems; therefore, improv-
Acinetobacter baumannii
ing the spectrum of action of antibiotics could be necessary to reverse this situation. Amikacin and silver salts
Amikacin
have well-known antimicrobial properties. However, both drugs lost their effectiveness against some bacte-
Aminoglycoside TagedEn
ria, such as Acinetobacter baumannii. This work aims to develop a nanodrug from silver nanoparticles (AgNPs)
functionalized with Amikacin against multidrug-resistant Acinetobacter baumannii.
Methods: AgNPs were produced using the bottom-up methodology and functionalized with Amikacin modi-
fied by the carbodiimide-based chemistry, forming AgNPs@Amikacin. Susceptibility tests were performed
using Amikacin-resistant Acinetobacter baumannii strains to assess the bacteriostatic and bactericidal poten-
tial of the developed nanodrug. The clinical strains were induced to form a biofilm, and biomass quantifica-
tion and the metabolic activity were determined.
Results: The AgNPs have a hydrodynamic diameter of the particles with a bimodal distribution, with a size of
37.84 nm. The FT-IR spectrum of AgNPs@Amikacin exhibits vibrational modes corresponding to Amikacin,
confirming the conjugation to AgNPs. Susceptibility testing demonstrated a minimal inhibitory and bacteri-
cidal concentration of < 0.5 mg/mL. The AgNPs@Amikacin reduced the biofilm metabolic activity of Acineto-
bacter baumannii at rates ≥ 50%, characterized by the minimal biofilm inhibition concentrations.
Conclusions: Results demonstrate a promising development of a new nanodrug with lower concentrations,
less toxicity, and greater efficacy against multidrug-resistant Acinetobacter baumannii.
© 2023 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All
rights reserved.TagedEn

TAGEDH1INTRODUCTIONTAGEDN emergence of treatment-resistant pathogens. It is estimated that by

2050, multidrug-resistant (MDR) microorganisms will cause devas-
TagedPThe success of antimicrobial therapy has been threatened and the tating damage to the health of the world population and the econ-
main reason is the indiscriminate use of antibiotics, which increases omy, with an economic impact of approximately US$ 100 trillion if
bacterial selective pressure. After several decades of successful measures to curb the emergence of MDR microorganisms are not
antimicrobial therapy, humanity faces a troubling prospect: the taken.1 In addition, the respiratory syndrome pandemic caused by
the Coronavirus (COVID-19) may anticipate this estimate. This fact is
related to azithromycin, an antibiotic of the class of protein synthesis
TagedEn * Address correspondence to Maiara L. Castilho, PhD, IP&D (Lab. 28), Av. Shishima inhibitor macrolide, that has been widely used in the prophylaxis of
Hifumi, 2911 - Urbanova, S~  dos Campos/SP, 12244-000, Brazil.
ao Jose
co-infections with the disease caused by Coronavirus, increasing the
E-mail address: mcastilho@univap.br (M.L. Castilho).
TagedEnConflict of interest: None to report. selective pressure of bacteria and being one of the main causes of the
TagedEnAuthor contributions: L.O.C., conceptualization and design; acquisition, analysis, emergence of MDR microorganisms.2 In a retrospective observational
and interpretation of data for the work; and writing (original draft); I.F., acquisition, study carried out at a university hospital in Terni (Umbria/Italy), an
analysis, and interpretation of data for the work; T.S.V., acquisition, analysis, and inter- increase in the incidence of patients colonized by carbapenem-resis-
pretation of data for the work; L.R., conceptualization, acquisition, analysis, and inter-
tant enterobacteria was observed, which increased from 6.7% in 2019
pretation of data for the work; M. L. C., conceptualization, design, analysis,
interpretation of data for the work, validation, supervision, and writing (original draft). to 50% in March-April of 2020.3 Although statistics on the impact of

https://doi.org/10.1016/j.ajic.2022.12.009
0196-6553/© 2023 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

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TagedEn872 L.d.O. Camargo et al. / American Journal of Infection Control 51 (2023) 871−878

the COVID-19 pandemic on antimicrobial resistance are still scarce in the first reaction step for forming the Amikacin-LA complex, LA was sol-
the world, data from the Central Laboratory in Southern Brazil dem- ubilized in a methanol and PBS buffer (pH 6.2) mixture at a 1:1 ratio.
onstrate a 130% increase in identifying Acinetobacter baumannii MDR Of this solution, 308 mL was added to the 990 mL of sulfo-NHS and the
strains comparing the first quarters of 2019 and 2021.4 Another study 900 mL of EDC. This solution was stirred for 30 minutes at 150 rpm. In
reports a detailed investigation of a carbapenem-resistant Acineto- the second step, 1050 mL of 0.85 mM Amikacin was added to the solu-
bacter baumannii outbreak in a community hospital’s COVID-19 tion, and the pH was corrected to 7.0 with the addition of 0.5 M NaOH to
intensive care units (ICU) that had not been previously identified in increase the activation effectiveness. In the end, an analysis is done in
this ICU the years before 2020. The infections caused by resistant UV-Visible to assess the effectiveness of the reaction, and then the pro-
A. baumannii are becoming more common in hospital settings and cess of purification of the synthesis begins. This step was performed by
present a significant clinical challenge as treatment choices for these dialysis with a membrane with a 0.5 kDa porosity (Repligen 131090T)
organisms are limited.5TagedEn until all synthesis by-products were eliminated and the characteristic
TagedPIn an attempt to reverse this problem, numerous studies have band of Amikacin-LA complex formation was observed in the UV-Visible
been carried out to improve the spectrum of action of antibiotics. spectrum. The data were plotted with the aid of the OriginPro version
Thus, developing drugs with new mechanisms of action capable of 8.5.1 program.TagedEn
blocking the bacterial resistance machinery is essential to ensuring
the health of humanity. Amikacin is an aminoglycoside capable of TagedH2Amikacin functionalization to silver nanoparticleTagedEn
inhibiting bacterial protein synthesis and is widely used against bac-
teria resistant to carbapenems.6 However, its activity is compromised TagedPThe AgNPs@Amikacin was synthesized using the Amikacin-LA
due to resistance mechanisms developed by bacteria, and aminogly- complex, which was functionalized into the surface area of AgNPs.
coside-modifying enzymes are the main defense tool.7 This resistance The Amikacin-LA complex (8 mg/mL) was added to AgNPs (4.5 £ 1012
has been observed in strains of Acinetobacter baumannii, a gram-neg- particles/mL) in the volume necessary to carry out the susceptibility
ative nosocomial bacillus that causes hospital infections, sepsis, and experiments; both were kept under agitation for 24 hours. The quan-
urinary infections in immunocompromised patients with a fatality tification and characterization of the nanodrug were performed using
rate greater than 50%.8TagedEn the analysis of UV-visible spectroscopy, DLS, and FT-IR spectroscopy.TagedEn
TagedPSilver has been used since antiquity for its bactericidal properties,
and with the advent of technology, nanostructures with this metal TagedH2ATR-FT-IR spectroscopyTagedEn
have shown an improvement in their activity due to their highly
reactive multifaceted structure. Silver ions are highly reactive and TagedPFourier transform infrared spectroscopy (FT-IR) analysis was per-
bind to bacterial proteins, causing structural changes in the cell wall formed using the FT-IR Spectrum 400 Spectrometer (Perkin-Elmer,
and membrane. Furthermore, they denature the DNA and ribosomal USA) with attenuated total reflectance (ATR). The ART has a dominant
proteins that synthesize substances essential for the metabolism of sampling technique in IR spectroscopy to analyze materials. The ART-
microorganisms. These effects are potentiated by the reactive oxygen FT-IR spectra were measured in the range of 4000 to 900 cm 1 with
species generated by the silver nanoparticles, which justifies its prob- 4 cm 1 spectral resolution and 16 scans in the transmittance mode.
able deleterious mechanism. 9 In an effort to develop a nano drug to The sample was placed directly on the ART crystal and dried to form
improve the harmful capacity of Amikacin and reduce its adverse a thin film. OPUS and Origin Pro version 8.5 were used for statistical
effects, the present study describes the antibiotic functionalization analysis.TagedEn
on synthesized silver nanoparticles. The effectiveness of the nano-
drug will be evaluated through susceptibility tests and biofilm inhibi- TagedH2UV−visible spectroscopyTagedEn
tion against resistant strains of Acinetobacter baumannii.TagedEn
TagedPUV−visible spectra were acquired using a DeNovix DS-11 (DeNo-
TAGEDH1METHODSTAGEDN vix Inc., USA) spectrophotometer in the spectral region 190−840 nm
with a resolution of 1 nm and an optical path length of 1 cm. The
TagedH2Synthesis of the silver nanoparticleTagedEn sample was deposited on the instrument pedestal for analysis.TagedEn

TagedPUsing the bottom-up methodology, AgNPs were synthesized from TagedH2Dynamic light scattering (DLS)TagedEn
the reduction of silver ions by borohydride, based on the methodol-
ogy described by Solomon et al. (2007).10 Reactant concentrations TagedPDynamic Light Scattering analysis was used to estimate the par-
were calculated based on the stoichiometric ratio of 2 parts borohy- ticles’ average hydrodynamic diameter and determine the colloidal
dride to one-part silver nitrate. This way, a solution of 30 mL of solution’s stability. A volume of 400 mL of the colloidal solution added
sodium borohydride contained in a 100 mL Erlenmeyer flask was to the polystyrene cuvette (Model ZEN0118, Sarstedt) is being mea-
cooled in an ice bath and placed on a magnetic plate with constant sured by the ZetaSizer nano equipment, ZS90 (Malvern), in “Size”
stirring. Reducing system energy induces the formation of smaller mode to determine the hydrodynamic diameter. An average of 3
particles, so the synthesis process takes place at low temperatures. analyses per sampling was performed to obtain the result, and with
After 35 minutes, 10 mL of silver nitrate solution was added to the the aid of the OriginPro version 8.5.1 program, the exported data
reducing agent, and as the silver nitrate was reduced, the solution were plotted.TagedEn
turned yellowish, indicating the formation of silver nanoparticles. TagedPThe stability of the nanostructured material was determined in
The AgNPs were characterized by UV-visible Spectroscopy and triplicate by analyzing the Zeta potential using the equipment Zeta-
Dynamic Light Scattering (DLS).TagedEn Sizer nano, ZS90 (Malvern), in “Zeta” mode. The colloidal solution
was added to the capillary cuvette with the electrode (model
TagedH2Chemical modification of amikacinTagedEn DTS1070, Malvern) using a syringe to avoid forming air bubbles.TagedEn

TagedPThe activator of the carboxylic group of alpha-lipoic acid (LA) used in TagedH2X-ray diffraction (XRD) analysisTagedEn
an aqueous medium was N-(3-dimethylaminopropyl)-N’-ethylcarbodii-
mide (EDC), which was used together with the reagent N-hydroxysucci- TagedPThe crystalline structure of AgNPs was identified using X-ray dif-
nimide (sulfo-NHS), a stabilizer that increases the process efficiency. In fraction (XRD-6000, Shimadzu). The thin film of AgNPs coated the

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TagedEn
Table 1 post-incubated agar plate observation for the presence or absence of
Antibiotic susceptibilities of A. baumannii clinical strains expressed as MIC values and bacteria.TagedEn
classification according to the clinical breakpoints defined by EUCAST 2022.

Antibiotics Clinical strains TagedH2AgNPs@Amikacin effect against the biofilm form of A. baumanniiTagedEn
AB 1 AB 2 AB 3
TagedPBiofilms were formed for 48 h on the bottom of a 96-well micro-
MIC SR MIC SR MIC SR plate using 107 UFC/mL of A. baumannii clinical strains in BHI broth at
(mg/mL) (mg/mL) (mg/mL)
approximately 35 °C. Then, the broth was removed, and the wells
Amikacin >16 R >32 R >32 R were washed twice with sterile saline solution of 0.9%. Different con-
Ciprofloxacin >2 R >2 R >2 R centrations of AgNPs@Amikacin were added to each well. The nega-
Gentamicin >8 R >8 R >8 R
Imipenem >8 R >8 R >8 R
tive control and growth control groups were also included, using the
Levofloxacin >4 R >4 R >4 R nanodrug and sterile saline solution of 0.9%, respectively. After incu-
Meropenem 32 R 32 R >32 R bation for 24 hours at 35 °C, the microplate was gently washed twice
R, resistant; S, susceptible; SR, classification according to EUCAST 2022 definitions. with sterile saline solution of 0.9%, and the biomass quantification
and metabolic activity were determined.TagedEn

TagedH2Biomass quantification by crystal violet assayTagedEn

XRD aluminum grid, and the spectrum was recorded with a CuKa TagedPIn order to quantify the biofilm biomass of A. baumannii, the crys-

radiation (λ = 1.54 A) in the 2u range from 35° to 80°.TagedEn tal violet assay was performed. After the content of each well was
removed and gently washed twice with a sterile saline solution of
TagedH2Bacterial strains and growth conditionsTagedEn 0.9% to remove planktonic and loosely bound cells, 200 mL of metha-
nol was added for 20 minutes to fix the biofilm. After stained with 1%
TagedPThe A. baumannii ATCC 19606 strain and the 3 clinical strains (wt/vol) crystal violet solution for 5 minutes at room temperature,
used, were provided by the Oswaldo Cruz Laboratory of S~ao Jose  dos the excess stain was washed off with sterile saline solution 0.9%. The
Campos (Brazil) through an agreement. The susceptibility profile and biofilms were resuspended in 200 mL of acetic acid and measured at
identification of clinical strains were confirmed by an automation A570 in SpectraCountTM (BS10001, Packard BioScience Company) to
system in the BD Phoenix equipment (Table 1), whose interpretation determine the biofilm biomass.TagedEn
of the minimum inhibitory concentrations is in accordance with the
standards standardized by the European Committee on Antimicrobial TagedH2Assessment of metabolic activity by MTT colorimetry assayTagedEn
Susceptibility Testing 2022 (EUCAST 2022).TagedEn
TagedPThe strains were stored in the freezer at -80°C in an enriched liq- TagedPTo determine the metabolic activity of microbial cells inhabiting
uid medium (BHI, Brain Heart Infusion) containing 20% glycerol in the biofilm population, the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
microtubes. For the reactivation, the strains were seeded in BHI broth Diphenyltetrazolium Bromide) colorimetric assay was used. After the
at 35°C for 24 hours and characterized as “pre-inoculum.” Subse- treatment with AgNPs@Amikacin, biofilms were washed twice with
quently, they were seeded by exhaustion in a selective solid medium, sterile saline solution of 0.9% to remove planktonic and loosely bound
such as MacConkey agar, and incubated for 24 hours. After this cells, and then 200 mL of MTT solution (0.25 mg/mL) was added for
period, the purity degree of the colonies formed was visually 1 hour at 35°C. After this period, the MTT solution was removed from
checked, and the inoculum was prepared.TagedEn the wells, and 200 mL of DMSO was added to dissolve the formed for-
mazan crystals. The absorbance of the solution was measured at
TagedH2Antibacterial activity of AgNPs@Amikacin in A. baumannii strainsTagedEn A570 nm using a SpectraCountTM spectrophotometer (BS10001,
Packard BioScience Company), and the minimal biofilm inhibition
TagedPThe antibacterial activity of AgNPs@Amikacin was determined concentration (MBIC50) was defined as the lowest drug concentration
using the broth microdilution method under the guidelines of ISO that prevented half of the maximal inhibitory concentration.TagedEn
20776-1 (International Organization for Standardization). Briefly, in
96-well microplates, 100 mL of Mueller-Hinton culture medium TagedH2Statistical analysisTagedEn
(HiMediaÒ , Mumbai, India) were added to each well. Serial fold dilu-
tions of AgNPs@Amikacin (the initial concentration of the nanodrug TagedPData were statistically analyzed using the GraphPad Prism 8.0.1
in the first well was 4.0 mg/mL, followed by 3 dilutions until the final software. A normality assessment was performed to define the
concentration of 0.5 mg/mL) and the antibiotic were made in the appropriate type of test. The ANOVA One-Way method comple-
microplate. A. baumannii strain suspension (100 mL) containing mented by Tukey’s test was used for results that present a normal
approximately 1.0 £ 106 CFU/mL was added to all the wells. Sterility curve, considering significance to be 5% (p≤0.05).TagedEn
control and growth control were also included. The microplate was
incubated for 18 hours at 35 °C. After this period, the Minimum Inhib- TAGEDH1RESULTSTAGEDN
itory Concentration (MIC) analysis was performed by the turbidity
method and confirmed by an OD600 reading in the Synergy HT TagedPThe synthesized silver nanoparticle showed maximum absorption
Multi-Detection Microplate Reader spectrophotometer (BioTek at 393 nm in the UV-Visible spectra, characteristic of a spherical
Instruments, USA). The lowest concentration of the drug that did not structure (Fig 1A). Figure 1B shows the DLS measurements used to
show any visible growth was taken as the upper limit for the MIC determine the hydrodynamic diameter of the particles with bimodal
range. The experiment was carried out in duplicate.TagedEn distribution, with the Gaussian with the highest intensity of nanopar-
TagedPThe Minimal Bactericidal Concentration (MBC) was determined ticles having an average diameter of 37.84 nm; and a second popula-
after the MIC. From the 96-well microplates, an aliquot of 10 mL from tion having an average diameter of 1.49 nm. The stability of the
the non-growth samples was seeded on a BHI agar plate using the particles was confirmed by the zeta potential value, which was found
drop method and incubated for 24 h at 35°C. The MBC was defined as to be 37.9 mV (Fig 1C), whereas phase formation was observed
the lowest concentration that induced death, according to pre- and using XRD patterns (Fig 1D).TagedEn

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TagedFiur
TagedEn874 L.d.O. Camargo et al. / American Journal of Infection Control 51 (2023) 871−878

Fig 1. Silver nanoparticle characterization (A) UV-Visible spectrum; (B) Histogram of hydrodynamic size distribution; (C) Zeta potential analysis; (D) Representative X-ray diffrac-
tion profile of the thin film.TagedEn

TagedPThe nanodrug showed different characterizations of the bare TagedPThe antibiotic resistance mechanism belongs to the aminoglyco-
nanoparticles. The UV-Visible spectra of AgNPs@Amikacin appear to sides class, directly related to modifying enzymes. Therefore, the
be a combination of the absorption spectra of antibiotics and AgNPs, chemical modification of Amikacin via carbodiimide implies the
where the absorption at 265 nm corresponding to Amikacin and the binding of a ligand to the amino functional groups of the antibiotic,
surface plasmon resonance (SPR) of AgNPs form a broad absorbance causing the enzymes to lose their substrate. Table 2 shows the sus-
centered at 395 nm (Fig 2A). Figure 2B shows the increase in the ceptibility tests of the strains to the Amikacin-LA complex, in which
hydrodynamic size of the nanodrug, which is 2.99 nm, corresponding it is possible to observe that the resistance profile was maintained for
to the topographical area of Amikacin on the surface of the nanopar- all clinical strains. However, the standard strain with a sensitivity
ticles. This result confirms the presence of the aminoglycoside func- profile was changed to a resistant profile.TagedEn
tionalized on the surface of the AgNPs, which was corroborated with TagedPThe deleterious action of the nanopharmaceuticals (AgNPs@Ami-
UV-vis spectra, a small redshift in the maximum absorption of the kacin) was evaluated, which showed reductions of at least 64-fold
nanoparticles. The colloidal stability of AgNPs@Amikacin was mea- compared to the antibiotic, evidencing that the MIC is <0.5 mg/mL
sured by Zeta potential, and the value was -33.7 mV, indicating that (Table 2). The MBC exhibited a growth inhibition of standard and
they are quite stable for biological application even after functionali- clinical strains analyzed at the lowest concentration of AgNPs@Ami-
zation (Fig 2C).TagedEn kacin tested (<0.5 mg/mL), which defined the efficiency of the nano-
TagedPFigure 2D shows the Fourier Transform Infrared spectrum of Ami- pharmaceuticals in terms of the concentration that completely killed
kacin (gray line) compared to the nanodrug (black line). The AgN- the strains. The growth inhibition of the microorganisms analyzed
Ps@Amikacin spectrum exhibits vibrational modes corresponding to was evident in the two MIC, and MBC assays at all concentrations of
the Amikacin molecule being identified by the bands of 3352 cm 1, AgNPs@Amikacin tested. This demonstrates the bactericidal and bac-
2928 cm 1, 1644 cm 1, 1544 cm 1, 1148 cm 1, and 1042 cm 1, con- teriostatic action of the nanopharmaceuticals.TagedEn
firming the presence of the antibiotic in the surface area of AgNPs.TagedEn TagedPThe MIC values for AgNPs@Amikacin were determined using a
TagedPThe antibiotic susceptibility profile of A. baumannii clinical strains broth microdilution assay. Thus, the concentrations tested in the bio-
was determined with a panel of antibiotics representing different films did not show growth in the MIC assay, encouraging such con-
classes, considering the intrinsic resistance profile to ampicillin, centrations in the biofilm application for MBIC determination. The
amoxicillin-clavulanate, aztreonam, cefazolin, cephalothin, cefalexin, crystal violet (CV) assay compared different concentrations of AgN-
cefadroxil, cefotaxime, ceftriaxone, ertapenem, trimethoprim, tetra- Ps@Amikacin. These results indicated that nanopharmaceuticals did
cycline, and doxycycline, confirming the multi-resistant profile of the not reduce biomass (Fig. 3A, B, and C).TagedEn
bacteria in the study (Clinical Breakpoint Table 2022; EUCAST).TagedEn TagedPThe results of evaluating bacterial metabolic activity by the MTT
TagedPTable 2 shows the growth inhibition of different A. baumannii colorimetric assay are shown in Figure 3. The application of AgN-
strains at different concentrations of Amikacin, the Amikacin-LA Ps@Amikacin to the A. baumannii biofilm reduced mitochondrial
complex, and AgNPs@Amikacin. At ≤4 mg/mL concentrations, the activity by 50% in all clinical strains using the concentration of 4 mg/
Amikacin and the Amikacin-LA complex did not inhibit clinical mL, with a significant difference of P<0.0001. At the lowest concen-
strains growth, whereas AgNPs@Amikacin did.TagedEn tration of the nanodrug used, there was also an altered metabolic

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TagedFiur TagedEnL.d.O. Camargo et al. / American Journal of Infection Control 51 (2023) 871−878 875

Fig 2. Nanodrug AgNPs@Amikacin characterization (A) UV-visible spectra; the absorption at 265 nm corresponds to Amikacin, and the SPR at 395 nm is characteristic of AgNPs; (B)
Histogram of hydrodynamic size distribution; (C) Zeta potential analysis; (D) Fourier Transform Infrared spectrum of Amikacin (gray line) compared to the nanodrug (black line).TagedEn

action concerning the control group, showing a reduction of 16% (AB behavior was also justified by Jesus et al. (2018).12 The phase forma-
1, Fig 3D), 13% (AB 2, Fig 3E), and 23% (AB 3, Fig 3F), with a significant tion confirmed the face-centered cubic structure for the AgNPs from
difference of P<0.0001.TagedEn characteristic peaks, such as (111), (200), (220), and (311), deter-
mined in the XRD patterns (Fig 1D). The same phase formation was
TAGEDH1DISCUSSIONTAGEDN reported by Salunke and coauthors (2014), and XRD analysis con-
firmed the crystalline nature of the silver nanoparticles.13TagedEn
TagedPControlling the multidrug-resistant microorganisms has been a TagedPAfter conjugation with the aminoglycoside, the Zeta potential of
major challenge for the health of the world’s population. Thus, to AgNPs’ synthesis, was measured to assess the stability of the colloidal
reverse this problem, this study demonstrated the improvement of suspension. Initially, it was −37.9 mV and changed to −33.7 mV,
the spectrum of action of the antibiotic Amikacin, which belongs to ensuring the stability of the colloid against microbiological tests.
the class of aminoglycosides, using nanotechnology. The AgNPs syn- Values between -30 mV and +30 mV represent an increase in the
thesized show spectrum is characteristic of spherical nanoparticles inhibition of electrostatic repulsion, causing an agglomeration and
due to the single band of resonant oscillation of electrons on the sur- sedimentation of the solute.14 A similar performance was observed
face of the formed nanoparticle, which corroborates the studies by by Lopez-Carrizales et al. (2018), analyzing silver nanoparticles with
Agnihotri et al. (2014).11 However, the bimodal behavior presented an average hydrodynamic diameter of 39.25 nm and a zeta potential
in the DLS analysis is characteristic of the synthesis of AgNPs due to of -40.8 mV. However, after the direct combination with Amikacin,
the Ostwald ripening process, whose nucleation occurs from seeds the zeta potential dropped to -21.10 mV, becoming a less stable solu-
that grow at different times. Thus, at the end of the synthesis process, tion and favoring the aggregation of nanoparticles.15 These studies
some particles do not grow due to the formation of depletion layers also demonstrate the need for drug modification to favor conjugation
around them, generating 2 nanoparticle populations. The same with AgNPs to ensure sound synthesis. Chemical modification of the

TagedEnTable 2
Growth inhibition of different A. baumannii strains after 18 hours of antimicrobial susceptibility testing by broth microdilution at different concentrations of Amikacin, the Amika-
cin-LA complex, and AgNPs@Amikacin.

Amikacin (mg/mL) Amikacin-LA complex (mg/mL) AgNPs@Amikacin (mg/mL)

A. baumannii 4.0 2.0 1.0 0.5 4.0 2.0 1.0 0.5 4.0 2.0 1.0 0.5

ATCC 19606 - + + + + + + + - - - -
AB 1 + + + + + + + + - - - -
AB 2 + + + + + + + + - - - -
AB 3 + + + + + + + + - - - -
NOTE. Positive (+): turbidity indicating growth; Negative (−): no turbidity indicating absence of growth. (n=3).

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TagedFiurTagedEn876 L.d.O. Camargo et al. / American Journal of Infection Control 51 (2023) 871−878

Fig 3. Biofilm susceptibility test of A. baumannii multidrug-resistant after 24h growth in the presence of AgNPs@Amikacin. Biofilm biomass quantification in Clinical strains: (A) AB
1; (B) AB 2; (C) AB 3; metabolic activity in clinical strains (D) AB 1; (E) AB 2; (F) AB 3. ns: P>0.05; * P≤0.05; ** P≤0.01; ***P≤0.001 and ****P≤0.00001. Data are means § SD (n = 3).TagedEn

drug is necessary for binding to the silver nanoparticle. Amikacin was adenylation, methylation, or phosphorylation of the drug’s amino
linked to alpha-lipoic acid via the carbodiimide reaction, in which groups.7 Modifying Amikacin by adding alpha-lipoic acid promotes
amide bonds are formed by the reaction of alpha-lipoic acid’s carbox- the bond between the amino groups and the carboxylic group,
ylic acid group with Amikacin’s primary amine groups. The sulfur causing the enzymes to lose their substrate by not interacting with
bonds in LA promote the covalent bonding of the aminoglycoside to the amino group. Table 2 shows that different A. baumannii strains
AgNPs, synthesizing the nanopharmaceuticals.16TagedEn did not exhibit growth inhibition at Amikacin LA complex concen-
TagedPThe vibrational modes 3352 cm 1 and 2928 cm 1 correspond to trations ≤ 4 mg/mL, indicating that the resistance profile was main-
the O-H stretching and the stretching of the aliphatic group CH2, tained for all clinical strains. However, the standard strain with a
respectively.17,18 The bands found in the fingerprint region are also sensitivity profile was switched to a resistant profile. This fact is
attributed to the molecule’s functional groups, so the band at 1644 directly related to a possible modification of the drug, in which the
cm 1 refers to the C=O angular deformation, and the bands at 1544 free primary amines seem to have a fundamental role in the phar-
cm 1, 1148 cm 1, and 1042 cm 1 correspond to the axial strains of macological action, and the modification via carbodiimide of the
NH2, C-O, and C-N, respectively.17−19TagedEn molecule suggests that the conjugation does not interfere in
TagedPFatima et al. (2019), describing similar bands when characteriz- the action of aminoglycoside-modifying enzymes, maintaining the
ing the aminoglycoside antibiotic, clearly indicate the drug’s profile of antibiotic resistance. Furthermore, the change in the sus-
authenticity, which corroborates the spectrum shown in ceptibility profile of the standard strain suggests that the amino
Figure 2D.18 The nanodrug spectrum shows vibrational modes that acid bond sites are essential for the pharmacological action of the
evidence the formation of the Amikacin-LA complex, which corre- antibiotic.TagedEn
sponds to the amide bond formed between the antibiotic molecule TagedPAn antibiotic is considered bactericidal when the MBC is less than
and alpha-lipoic acid. Castilho and coauthors (2017) describe the 4 times the MIC value.23 Agents with antibiotic properties can have a
region from 1020 to 1250 cm 1 as referring to the amide bond for- deleterious effect or limit bacterial growth. Bactericidal drugs have
mation attributed to the axial deformation of C−N.16 Thus, the better activities at lower therapeutic dosages, inhibiting the microor-
bands’ increased intensity of 1104 cm 1, 1232 cm 1, and 1260 cm-1 ganism’s recrudescence and the emergence of resistance mecha-
in the nanodrug spectrum is attributed to the formation of amide nisms.13 Although there are several studies on the antimicrobial
III, confirming the formation of the Amikacin-LA complex.16,20,21 action of silver nanoparticles, either in isolation or associated with
The bending vibrations of N-H are found in the region of 1474 cm-1, antibiotics against different microorganisms, research associating
coming from the amide bond between Amikacin and alpha-lipoic Amikacin with AgNPs against resistant strains of A. baumannii is rare.
acid, in which there is a difference in band intensity between Ami- Furthermore, the characteristics of each nanoparticle are quite pecu-
kacin’s spectra and AgNPs@Amikacin.22TagedEn liar, and the different methodologies tested make it difficult to com-
TagedPThe 1544 cm 1 band is attributed to the NH2 vibrational mode, pare the results. Studies carried out by Kaur et al. (2019) using silver
the primary amine present in the antibiotic molecule. There is a loss nanoparticles with an average hydrodynamic diameter of 61 nm,
of intensity in this band in the spectra of the nanodrug, which eviden- linked to Amikacin through hydrogen bonds with the polymer poly-
ces the chemical modification via carbodiimide of the Amikacin to vinylpyrrolidone, proved to be efficient against Escherichia coli
form the complete one, allowing its binding in the surface area of the strains. Although the susceptibility test was performed by disk diffu-
nanoparticles. At 1574 cm 1, it corresponds to the carbonyl group sion, the increase in the halo of inhibition proved to be a promising
(C=O) and to the stretching of N-H, vibrational modes that are modi- association.24 Despite the loss of stability of the colloidal system pro-
fied with the amide bond and present with a band of great intensity posed by Lopez-Carrizales et al. (2018), silver nanoparticles associ-
in AgNPs@Amikacin’s spectra.17−19,21TagedEn ated with Amikacin reduced the MIC in susceptibility tests against
TagedPThe main resistance mechanism developed by A. baumannii A. baumannii strains from 128 to 4 mg/mL. This is because, after asso-
strains against aminoglycosides is the enzymatic modification of ciation, the mean size of AgNPs changed to 947 nm.15 ºysakowska

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 TagedEnL.d.O. Camargo et al. / American Journal of Infection Control 51 (2023) 871−878 877

et al. (2015) also demonstrated the antimicrobial activity of silver nanoparticles from 10 to 50 nm in diameter against multidrug-resis-
nanoparticles against MDR Acinetobacter spp. The MIC was 0.78 mg/ tant A. baumannii biofilms. Analyzing the bacteria’s virulence genes,
ml, using a colloidal suspension of silver nanoparticles with a mean the authors observed that 25 mg/mL of AgNPs could inhibit the for-
diameter between 2 and 100 nm. Although the analyses were carried mation of biofilms by inactivating the Bap, OmpA, and CsuA/B genes.
out only with the planktonic strains, the nanoparticles seem promis- The expression of genes responsible for bacterial virulence was also
ing in the topical treatment of wounds contaminated by MDR inhibited, reducing the pathogenicity of the microorganism and
microorganisms.25TagedEn increasing susceptibility to colistin.32TagedEn
TagedPGram-negative bacteria are more resistant to pharmacological
action due to the external phospholipid membrane that hinders the
TAGEDH1CONCLUSIONSTAGEDN
permeability of antimicrobials.7 One of the possible justifications for
the synergism of the association between the silver nanoparticle and
TagedPThe process of synthesizing silver nanoparticles from the oxida-
Amikacin is the favoring of the antibiotic’s passage through cell mem-
tion of silver nitrate using sodium borohydride was well character-
branes, considering the metal’s affinity for phosphorus, which causes
ized as to the size and stability of the resulting colloidal suspension.
the instability of the phospholipid layer. Another fact is that silver
The specific bands of amides and carbonyls referring to Amikacin’s
binds to sulfur-containing compounds, such as the amino acids
chemical modification were demonstrated, as well as the characteris-
methionine and cysteine found in DNA and proteins. This addition
tic functional groups of the drug, confirming the effectiveness of the
alters bacterial metabolism, which is also affected by highly reactive
synthesis of the AgNPs@Amikacin. Susceptibility testing demon-
free oxygen species.26 Oxidative stress caused by the presence of sil-
strated a minimal inhibitory and bactericidal concentration of < 0.5
ver can inhibit the action of aminoglycoside-modifying enzymes,
mg/mL of AgNPs@Amikacin. These results demonstrate how effective
favoring the action of Amikacin in inhibiting protein synthesis by
the deleterious action of the nanodrug is. In addition, the low MIC
binding to the 30S ribosomal subunit (9). These probable mecha-
concentration of the nanodrug may also be associated with the inhi-
nisms of action justify the total inhibition of the growth of resistant
bition of the action of aminoglycoside-modifying enzymes, reversing
strains of A. baumannii tested at a minimum inhibitory concentration
the mechanisms of bacterial resistance. The AgNPs@Amikacin
and minimum bactericidal concentration < 0.5 mg/mL of Amikacin
reduced the metabolic activity of the biofilm of multidrug-resistant A.
bound to silver nanoparticles with an average hydrodynamic diame-
baumannii by ≥ 50% inhibition with exposure to 4 mg/mL, attesting to
ter of approximately 37 nm. Thus, when biofilm cells of the same
the efficiency of the new treatment. These results are promising for
strain were tested, MBECs increased dramatically relative to the
developing a new nanodrug with lower concentrations of Amikacin
planktonic MICs.27TagedEn
and, consequently, less toxicity and greater efficacy against multi-
TagedPBiofilm is a bacterial association in a polymer matrix composed
drug-resistant A. baumannii.TagedEn
mainly of polysaccharides that protect them against the host’s
immune system and the action of antibiotics. Thus, CV assays showed
that A. baumannii biofilms exhibited a resistance profile to AgNPs@A- TagedH1AcknowledgmentsTagedEn
mikacin. This fact could be related to the positive charge of dye,
which binds to negatively charged components such as polysacchar- TagedPThe authors would like to thank the support granted by FAPESP
ides, proteins, or nucleic acids. Therefore, the increase in biomass (Project 2018/23898-6) and CNPq (Projects 427602/2018-1 and
may be related to the type of death caused by the AgNPs@Amikacin, 302994/2018-4).TagedEn
which may have caused an increase in negatively charged residues,
increasing the formation of the complex with the dye, giving a false
interpretation of the increase in biomass.28 The antibacterial activity TagedH1ReferencesTagedEn
of the nanodrug in planktonic form proved to be quite effective, but
TagedP 1. O’Neill J. Review on antimicrobial resistance: tackling drug-resistant infections
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attributed to the impedance created by the biofilm matrix, which TagedP 2. Falavigna M, Colpani V, Stein C, et al. Guidelines for the pharmacological
treatment of COVID-19. The task-force/consensus guideline of the Brazilian
considerably protects bacteria from antibiotic agents.29 Although the
association of intensive care medicine, the Brazilian society of infectious dis-
CV assay’s analysis has not shown an effective inhibition of biofilm, eases and the Brazilian society of pulmonology and tisiology. Rev Bras Ter
the MBIC50 formed by the clinical strains of A. baumannii was defined intensiva. 2020;32:166–196.TagedEn
as the concentration that resulted in ≥ 50% inhibition of the meta- TagedP 3. Tiri B, Sensi E, Marsiliani V, et al. Antimicrobial stewardship program, COVID-19,
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TagedEn878 L.d.O. Camargo et al. / American Journal of Infection Control 51 (2023) 871−878

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