Microbial Pathogenesis 146 (2020) 104249

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Inhibition of biofilm and virulence properties of Pseudomonas aeruginosa by T

sub-inhibitory concentrations of aminoglycosides
Fazlurrahman Khana, Jang-Won Leeb, Aqib Javaidc, Seul-Ki Parka, Young-Mog Kima,b,∗
a
Institute of Food Science, Pukyong National University, Busan, 48513, South Korea
b
Department of Food Science and Technology, Pukyong National University, Busan, 48513, South Korea
c
Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, 201306, UP, India

A R T I C LE I N FO A B S T R A C T

Keywords: Aminoglycosides are a commonly used class of antibiotics; however, their application has been discontinued due
Aminoglycosides to the emergence of multi-drug resistance bacterial strains. In the present study, the subinhibitory concentrations
Antibiofilm (sub-MIC) of several aminoglycosides were determined and tested as an antibiofilm and for their anti-virulence
Anti-virulence properties against Pseudomonas aeruginosa PAO1, which is an opportunistic foodborne pathogen. P. aeruginosa
LasR
PAO1 exhibits multiple mechanisms of resistance, including the formation of biofilm and production of several
Molecular docking
Pseudomonas aeruginosa
virulence factors, against aminoglycoside antibiotics. The sub-MIC of these antibiotics exhibited biofilm in-
hibition of P. aeruginosa in alkaline TSB (pH 7.9). Moreover, various concentrations of these aminoglycosides
also eradicate the mature biofilm of P. aeruginosa. In the presence of sub-MIC of aminoglycosides, the mor-
phological changes of P. aeruginosa were found to change from rod-shaped to the filamentous, elongated, and
streptococcal forms. Similar growth conditions and sub-MIC of aminoglycosides were also found to attenuate
several virulence properties of P. aeruginosa PAO1. Molecular docking studies demonstrate that these ami-
noglycosides possess strong binding properties with the LasR protein, which is a well-characterized quorum-
sensing receptor of P. aeruginosa. The present study suggests a new approach to revitalize aminoglycosides as
antibiofilm and antivirulence drugs to treat infections caused by pathogenic bacteria.

1. Introduction
Pseudomonas aeruginosa is a pathogenic bacterium that has effec-
tively emerged as a cause of infections in human hosts via its biofilm
lifestyle [1]. Further, it has been identified as a nosocomial biofilm-
forming pathogen that causes chronic infections in the lungs of patients
with cystic fibrosis [2] and is related to infections that are associated
with otitis and burn wounds [3,4]. The formation of a biofilm an as-
semblage of bacterial cells that is encased by a self-produced structure
that comprises extracellular polymeric substances (EPS), such as exo-
polysaccharides, protein, and extracellular-DNA (e-DNA) is well-re-
cognized as a common resistance property against antibiotics and ad-
verse environmental conditions [5,6]. Biofilm formation by P.
aeruginosa induced major chronic infections in humans and animals and
constitutes a bacterial mechanism that allows resistance against mul-
tiple types of conventional antibiotics, including aminoglycosides
[7–9]. In P. aeruginosa, biofilm formation is also associated with the
production of multiple virulence factors that serve vital roles in bac-
terial pathogenesis and survival [10–13]. Furthermore, recent studies
have found that targeting these virulence factors enables the use of
conventional antibiotics and the simultaneous reduction of their se-
lective pressure for resistance mutations [14–16]. Thus, attenuation of
virulence properties, along with the inhibition of biofilm formation of
P. aeruginosa, is a promising therapeutic strategy, as evidenced by
several previous studies [17,18]. Although several potential drugs that
act as antibiofilm, antibacterial, and antivirulence agents have been
either isolated from biological sources or chemically synthesized, more
drugs need to be discovered [19–27].
Aminoglycosides are a commonly used class of conventional anti-
biotics against Gram-negative bacteria, along with β-lactams and
fluoroquinolones [28]. Aminoglycosides were previously reported to be
limited in their penetration through the biofilm matrix and cell wall
and are encountered by modifying enzymes and efflux pumps [29–33].
Furthermore, the misuse and overuse of aminoglycosides over the past
decades have resulted in resistance development via several means,
including enzymatic modification (i.e. acetyltransferases, phospho-
transferases, and nucleotidyltransferases), binding target modification,
reduced drug permeability, and efflux system [31,34–36]. Currently,

∗
Corresponding author. Department of Food Science and Technology, Pukyong National University, Busan, 48513, South Korea.
E-mail address: ymkim@pknu.ac.kr (Y.-M. Kim).

https://doi.org/10.1016/j.micpath.2020.104249
Received 30 January 2020; Received in revised form 19 March 2020; Accepted 4 May 2020
Available online 11 May 2020
0882-4010/ © 2020 Elsevier Ltd. All rights reserved.
F. Khan, et al.
Fig. 1. Chemical structure of the aminoglycosides tested in the present study.
though fluoroquinolone is commonly used to treat P. aeruginosa, several
reports demonstrate that the P. aeruginosa bacteria exhibit enhanced
biofilm formation and production of virulence factors in the presence of
the drug [37,38]. Therefore, expanding the variability of drugs options
and targets is highly important for the sustainable use of conventional
antibiotics in the future [39,40].
As such, in the attempts to revitalize drug action, several alternative
strategies have been developed over the past few decades [41,42]. One
of these strategies is to modify the culture environment to become more
alkaline, which was particularly effective in enabling gentamicin, to-
bramycin, and streptomycin to exhibit antibiofilm action against P.
aeruginosa [43,44]. Due to their similarity in structure, several other
aminoglycosides [e.g. amikacin, gentamicin, kanamycin, neomycin B,
paromomycin, and netilmicin (chemical structures presented in Fig. 1)]
have been selected in the present study to evaluate their antibiofilm and
antivirulence activities under an alkaline environment (pH 7.9). Fur-
thermore, in vitro phenotypic studies were also validated by molecular
docking to elucidate quorum-sensing (QS) suppression mechanisms
using these aminoglycosides. From the present study, considerable in-
sight is gained on (1) changes in biofilm architecture and biofilm-
forming cellular morphology upon exposure to drugs, as revealed by
microscopic assays, and (2) drug anti-virulent activities by their inter-
actions with QS signal molecules (i.e. LasR), as revealed by a molecular
docking assay.
2
Microbial Pathogenesis 146 (2020) 104249
2. Materials and methods
2.1. Bacterial strains, culture conditions and chemical reagents
The human pathogenic bacterium Pseudomonas aeruginosa PAO1
KCTC1637 was obtained from Korean Collection for Type Cultures
(KCTC, Daejeon, Korea). The growth condition and culture media for
this strain was followed according to our recent report [45,46]. These
bacterial cultures were allowed to grow in tryptic soy broth (TSB; Difco
Laboratory Inc., Detroit, MI, USA) with pH of 7.9. The growth condition
of these bacterial strains was 35 °C temperature under aerobic condi-
tion. The aminoglycoside antibiotics such as amikacin, gentamicin,
kanamycin, neomycin, netilmicin, and paromomycin aminoglycoside
antibiotics were purchase from Sigma-Aldrich Co. (St. Louis, MO, USA).
The stocks of each antibiotic were prepared in acidified water (pH 5.9).
2.2. Minimum inhibitory concentration (MIC)
The methodology that was employed to determine the MIC of each
aminoglycoside against P. aeruginosa was adapted from a previous re-
port [44,47,48]. Briefly, the MIC determination was performed by in-
cubating the bacterial seed culture (with an initial OD of 0.05) with
different concentrations of each antibiotic. The cell culture (240 μl) was
placed in a 96-well microtiter plate, which contained different con-
centrations of aminoglycosides. The plate was kept under shaking
conditions (567 cycles per min) in a microplate reader for 24 h at 35 °C.
At the end of the incubation period, the optical densities of the bacterial
cells were measured using the microplate reader (BioTek, Winooski, VT,
USA) at a wavelength of 600 nm (OD600).
F. Khan, et al.
2.3. Biofilm formation assays
Crystal violet staining was used to evaluate biofilm formation, as
described in previous studies [45,49]. Briefly, the cell culture of the
bacteria with an initial OD of 0.05 was incubated with the sub-MIC of
each aminoglycoside in TSB (pH 7.9) media in a 96-well microtiter
plate. After 24 h of incubation at 35 °C, the number of total cells (i.e.
planktonic and biofilm cells) was enumerated by measuring the OD at
600 nm. After measuring the OD, the free-floating planktonic cells were
discarded and the adhered cells that were present in the titer wells were
washed three times with distilled water. Furthermore, the adhered cells
were stained with aqueous crystal violet (0.1%) and incubated for
20 min. After staining, the crystal violet solution was discarded from
each titer well and the stained cells were washed three times with
distilled water and air dried. Moreover, the dye-bound cells were wa-
shed three times with distilled water and solubilized using 95% ethanol.
The quantitative nature of the stained cells was measured using a mi-
croplate reader (BioTek, Winooski, VT, USA) at 570 nm.
In addition, the eradication effect of different concentrations
(above-MIC, MIC, and sub-MIC) of aminoglycosides on the mature
biofilm of P. aeruginosa was evaluated, as described in previous studies
[45,50]. Firstly, the cell culture (initial OD of 0.05) of P. aeruginosa was
placed in the 96-well microtiter plate to allow mature biofilm formation
at 35 °C for 24 h of incubation under non-shaking conditions. After
discarding the planktonic cells, the established mature biofilms in the
microtiter plate were washed twice with sterile TSB. The surface-at-
tached cells in every well were treated with different concentrations of
each aminoglycoside and incubated for 12 h at 35 °C. After incubation,
the free-floating planktonic cells were removed from each well and
washed three times with distilled water, followed by staining with
crystal violet (0.1%) for 20 min. The adhered cells that were stained
with crystal violet were washed three times with distilled water and air
dried. These stained cells were dissolved with 95% ethanol, and their
quantitative values were determined using a plate reader at 570 nm.
Each experiment of the biofilm assay was performed in triplicate.
2.4. Analysis of biofilm architecture using a scanning electron microscope
Changes in the biofilm architecture and cell morphology of P. aer-
uginosa in the presence of each aminoglycoside at sub-MICs were vi-
sualized using a scanning electron microscope (SEM) [44,50]. The SEM
samples of the biofilm cells were prepared according to the aforemen-
tioned procedure. The cells of P. aeruginosa were allowed to grow on the
surface of the nylon membrane (0.5 × 0.5 cm) in the presence of the
sub-MIC of aminoglycosides in a 24-well titer plate. The titer plate was
incubated for 24 h at 35 °C. After incubation, the cells were fixed by
adding formaldehyde and glutaraldehyde. The free-floating cells that
were present in the wells were carefully removed, while the attached
cells on the nylon membrane were gently washed three times using
phosphate buffered saline (pH 7.4). After washing, biofilm cells were
dehydrated by successive concentrations of ethanol (50, 70, 80, 95, and
100%) and freeze-dried using a freeze dryer (model no. FD8518, il-
ShinBiobase Co. Ltd., Korea). After fixing on the surface of the SEM
stubs, coating of the cells was carried out with white gold for 120 s
using an ion-sputter (E−1010, Hitachi, Tokyo, Japan) The biofilm cells
were then visualized using the JSM-6490LV microscope (JEOL, Tokyo,
Japan) at a magnification of × 5000 and a voltage of 15 kV.
2.5. Analysis of virulence factors
Quorum sensing (QS) regulates various virulence properties such as
pyocyanin production, pyoverdine production, and protease activity,
within P. aeruginosa and were determined in the presence of the sub-
MIC of aminoglycosides. The procedure used for analysis of virulence
activity was carried out according to previously reported methods
[44,51]. Pyocyanin production in the cells of the culture (incubated at
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Microbial Pathogenesis 146 (2020) 104249
35 °C for 12 h), which are treated with the sub-MIC of each ami-
noglycoside, was evaluated by colorimetric assays, as explained by
Essaar et al. [52]. The pink color that is obtained after the extraction
were quantified by OD measurement at 520 nm. The pyoverdine esti-
mation was based on the florescence measurement of the cell-free su-
pernatant, as explained in an earlier study [53]. The cell free super-
natant was obtained from the P. aeruginosa cell culture that was grown
overnight and treated with the sub-MIC of aminoglycosides and mea-
sured OD at 405 nm. LasA protease activity of the P. aeruginosa culture
that was treated with the sub-MIC of the aminoglycosides was de-
termined based on the digestion of azocasein, as described earlier [12].
The filtered supernatant was mixed with 0.3% azocasein and incubated
for 4 h at 4 °C to allow the digestion of azocasein. The undigested
azocasein precipitated with trichloroacetic acid (10%) and was then
centrifuged to obtain the cell-free supernatant. Furthermore, the su-
pernatant was treated with 1 M NaOH, followed by OD measurement at
440 nm.
2.6. Analysis of flagellar motility
The impact of the sub-MIC of all aminoglycosides on flagellar-
mediated motilities (e.g. swimming and swarming) in P. aeruginosa was
evaluated using a previously described method [12]. The swarming
motility was determined on the surface of semi-solid agar media with
the composition of 0.5% casamino acids, 0.5% glucose, and 0.4% Bacto
Agar, as prepared in the Luria Britani (LB) broth. Similarly, another
flagellar motility, i.e. swimming, was also performed in the solid agar
media, but with a different medium composition that consisted of 0.2%
casamino acids, 0.3% Bacto Agar, and 30 mM glucose. After auto-
claving these media, the sub-MIC of each aminoglycoside were added
and poured in Petri dishes to allow solidification. The P. aeruginosa cell
culture (5 μl) that was grown overnight was placed in the center of each
solid agar plate and incubated for 24 h at 35 °C. The swarming and
swimming motilities of the cells on the surface of the agar plate were
estimated by measuring the diameter (cm) of the traveled cells.
2.7. Molecular docking analysis
AutoDock MGL tools-4.0 was used for performing docking studies as
it is one of the most frequently used docking tools [54]. Molecular in-
teractive visualization and analysis of molecular structures were carried
out in BIOVIA Discovery Studio 2019 [55]. The 3-D structure of LasR
with 1.4A resolution was retrieved from the protein data bank (ID:
3ix3). Preprocessing of LasR, such as optimization, energy minimiza-
tion and addition of hydrogen atoms, was done by SPDBV 4.10 [56].
This involves removal of all the heteroatoms, its natural ligand i.e. 3-
oxo-dodecanoylhomoserine lactone (OdDHL) and coordinates added by
SPDBV (Swiss PDB viewer) software in the LasR pdb file. 2D ligand
structures were generated in ChemSketch by using corresponding Inchl
key values of ligands retrieved from Pubchem. Open Babel software was
used to convert ligand structures from 2D to 3D. During the format
conversion, in order to prevent any structural changes in the ligands,
the chiral centers of each ligand were selected in their original state.
Following active site residues were used for the generation of Grid box
Leu-36, Tyr-47, Tyr-56, Tyr-64, Asp-73, Thr-75, Ile-52, Ala-50, Gly-38,
Leu-39, Val-76, Cys-79, Trp-88, Tyr-93, Leu-110, Leu-125, Gly-126,
Thr-80, and Ser-125 with coordinates of X:8.833, Y:5.881, and Z:20.282
to specify the possible interaction area of ligands [57,58]. Each ami-
noglycoside was docked into LasR protein grid using Lamarckian ge-
netic algorithm. Also, 3-oxo-C12-HSL (OdDHL), a natural ligand of
LasR, was used as a positive control.
2.8. Statistical analysis
Each graph in the manuscript were plotted using GraphPad Prism
7.0 (GraphPad Software Inc., San Diego, CA). Furthermore, each data
F. Khan, et al.
Fig. 2. MIC determination of the aminoglycosides against P. aeruginosa PAO1. (A) Bactericidal effect of amikacin and netilmicin and (B) Bactericidal effect of
kanamycin, paromomycin, neomycin and gentamicin.
was also analyzed by performing one-way ANOVA followed by
Dunnett's posttest and the results were presented as means ± SD.
3. Results
3.1. Determining the minimum inhibitory concentrations of aminoglycosides
To determine the MIC and decide the sub-MIC of aminoglycosides,
an alkaline culture (TSB, pH 7.9) was used to grow P. aeruginosa.
Different concentration ranges of each aminoglycoside were used to
treat P. aeruginosa, as presented in Fig. 2A and B. Under the afore-
mentioned conditions, the MIC of aminoglycosides was identified as
2 μg/ml for amikacin and 4 μg/ml for netilmicin (Fig. 2A), and 128 μg/
ml for kanamycin, 128 μg/ml for paromomycin, 16 μg/ml for neo-
mycin, and 8 μg/ml for gentamicin (Fig. 2B). Subsequently, the sub-MIC
levels were chosen based on these results and hover below the MIC. The
sub-MIC of each aminoglycoside was used to study the biofilm inhibi-
tion and attenuation of the virulence properties of P. aeruginosa.
3.2. Biofilm inhibition and mature biofilm dispersion by aminoglycosides
The impact of the sub-MIC of each aminoglycoside on biofilm for-
mation in the TSB (pH 7.9) was evaluated. The results demonstrate that
each aminoglycoside inhibited the formation of biofilm, and inhibition
was not a concentration-dependent process (Fig. 3). As observed in
Fig. 3, the biofilm inhibitory effect varied at certain sub-MIC levels of
each aminoglycoside. However, examination of the biofilm cell and
architecture by SEM found that the morphology of the cells changed
with exposure to different sub-MIC levels of the aminoglycosides
(Fig. 4). Furthermore, aminoglycosides at different sub-MIC levels
caused different forms of morphology of P. aeruginosa. Various changes
in morphology were observed, including chain formation, elongation,
filamentation, and streptococcal form development. For instance, ami-
kacin at a high sub-MIC (1 μg/ml) shows filamentation (Fig. 4). Kana-
mycin also induced a slight elongation of the cells at a high sub-MIC
(64 μg/ml), whereas at a low sub-MIC (4 μg/ml), the cell completely
changed to the streptococcal form. In the presence of netilmicin ami-
noglycoside, the cells were found to change to the streptococcal form at
all tested sub-MIC levels. In the presence of a high sub-MIC (8 μg/ml)
for neomycin, the cellular morphology constituted the chain form,
whereas at a low sub-MIC (0.5 μg/ml), all bacterial cells were in the
streptococcal form. In the presence of paromomycin, the streptococcal
form was found at a low sub-MIC (32 μg/ml). Interestingly, upon ap-
plication of gentamicin at a high sub-MIC (1 μg/ml), the cells were
highly elongated; however, at a low concentration (0.25 μg/ml), only
rod-shaped morphology was observed. Thus, based on the SEM ana-
lysis, it is concluded that the value of biofilm formation is changed due
to changes in cellular morphology, and thus is independent of
4
Microbial Pathogenesis 146 (2020) 104249
concentration.
In other words, the formation of chain, elongation, and filamentous
morphology at a high sub-MIC results in a slightly higher value of
biofilm formation. The results demonstrate that the sub-MIC of each
aminoglycoside exhibits significant inhibition of biofilm formation. To
determine the dispersal of the P. aeruginosa mature biofilm by each
aminoglycoside, a mature biofilm was allowed to form for 24 h. The
mature biofilm was treated with different concentrations (above-MIC,
MIC, and sub-MIC) of each aminoglycoside and incubated for 12 h to
allow eradication. Results indicate that each aminoglycoside exhibits
significant dispersal of the mature biofilm (Fig. 5). The eradication
process was found to be dependent on concentration, as it is higher at
above-MIC levels and lower at sub-MIC levels. The concentration-de-
pendent eradication of biofilm may be due to the electrostatic inter-
actions between the positively charged amino group of the aminogly-
coside and the negatively charged component of the biofilm matrix.
3.3. Inhibition of virulence properties
Biofilm formation, along with most other virulence properties of P.
aeruginosa that emerge during biofilm formation, are regulated with
involvements by the QS signaling system. Thus, in the present study,
along with seeking to understand drug inhibitory activity against the
bacterial biofilm, we also sought to examine their effects on pyocyanin
production, pyoverdine production, and LasA protease activity.
Pyocyanin and pyoverdine are virulent factors that are produced by P.
aeruginosa and contribute to bacterial virulence and pathogenesis.
Results have shown that high sub-MIC levels of all tested antibiotics
effectively inhibited the synthesis of pyocyanin, while both high and
low sub-MIC concentrations of these drugs are effective in inhibiting
pyoverdine synthesis (Fig. 6A and B). Similarly, a high sub-MIC of each
aminoglycoside also reduces LasA protease activity significantly
(Fig. 6C). At the initiation of the biofilm formation process, P. aerugi-
nosa chemotactically moved to the biotic or abiotic surfaces and be-
came sessile in nature. In the present study, the flagellar motility, i.e.
swarming and swimming, was monitored in the presence of each ami-
noglycoside at their sub-MIC levels. Each aminoglycoside at a high sub-
MIC significantly attenuated swarming motility compared to in the
control group (Fig. 7). A similar finding was obtained regarding
swimming motility inhibition by all aminoglycosides, in which high
sub-MIC levels reduced bacterial motility (Fig. 8). Overall, both the
swimming and swarming motilities of P. aeruginosa were more effec-
tively inhibited by the high sub-MIC of all tested aminoglycosides.
3.4. Interaction of aminoglycosides with the LasR amino acid residue
The in-vitro anti-quorum-sensing-mediated inhibition of virulence
properties of P. aeruginosa was further validated using a molecular
F. Khan, et al. Microbial Pathogenesis 146 (2020) 104249

Fig. 3. Effects on biofilm formation and cell growth of P. aeruginosa PAO1 in the presence of sub-MIC of aminoglycosides. (A) Amikacin, (B) Gentamicin, (C)
Kanamycin, (D) Neomycin, (E) Netilmicin and (F) Paromomycin. *P < 0.05 and **P < 0.01 were accepted as statistically significant and ns indicated non-
significance.

Fig. 4. Microscopic visualization of biofilm architecture and cell morphology by SEM in the presence of different sub-MIC of aminoglycosides.

5
F. Khan, et al.
Fig. 5. Dispersion of established 24-h mature biofilm of P. aeruginosa PAO1 by
various concentrations of aminoglycosides. (A) Amikacin and netilmicin, (B)
Gentamicin and neomycin, and (C) Kanamycin and paromomycin. *P < 0.05
and **P < 0.01 were accepted as statistically significant and ns indicated non-
significance.
docking strategy. Results from the molecular docking of aminoglyco-
sides with LasR, amikacin, gentamicin, kanamycin, netilmicin, strep-
tomycin, and tobramycin revealed greater binding efficacy with the
active site that is present in LasR. A relatively higher score than that of
OdDHL was observed (Table 1). However, neomycin and paromomycin
exhibited slightly lower binding energy than OdDHL. The probable
reason for this binding efficiency difference involves the improved
hydrogen bonding and the presence of hydrophobic interactions of
these compounds with residues that are present in the active site of
LasR. Residues of LasR, including Tyr-56, Trp-60, Tyr-64, Asp-73, Arg-
61, Ser-129, Cys-79, Gly-126, Thr-75, Val-76, Thr-115, Trp-88, Ala-105,
Leu-125, Leu-40, Leu-39, Asp-65, and Glu-48, have been shown to be
involved in forming hydrogen bonds with different aminoglycosides
(Table 1).
6
Microbial Pathogenesis 146 (2020) 104249
Residues, such as Tyr-56, Trp-60, Asp-73, and Ser-129, were found
to be common in most interactions, which is consistent with the re-
sidues that are involved in the binding of OdDHL. Most compounds
interacted with LasR via a common residue, i.e. Trp-60, which seems to
be a potential drug target. However, this finding requires further ex-
perimental validation. Apart from hydrogen bonding, most compounds
exhibited different types of stacking interactions with the residues of
the active site of LasR, especially with aromatic amino acids, such as
Tyr-47 and Tyr-64. Further, this may be yet another reason for the large
negative values of the binding energy. The 2D- and 3D-docked con-
formation of each aminoglycoside into the active site of LasR, along
with electrostatic interactions, such as H-bonds, van der Waals forces,
Pi-Pi stacking, Pi-alkyl stacking, and amide-Pi stacking, are represented
in Fig. 9.
Based on the docking studies, the aminoglycosides can be concluded
to interact with the QS receptor LasR. Based on the molecular docking
studies, these aminoglycosides can be used as potential antibiofilm and
virulence-attenuating drugs against P. aeruginosa.
4. Discussion
Currently, alternative strategies have considered revitalizing these
drugs using their sub-MIC levels, shifting to other targets that are clo-
sely associated with biofilm formation, which includes the quorum-
sensing system and their down-regulated virulence factors, and using
various drug combinations (i.e. combinatory strategy) [7,15,59]. The
present study was designed to revitalize aminoglycosides as antibiofilm
and virulence-attenuating drugs against P. aeruginosa. Furthermore, due
to previous evidence that suggests the supportive role of an alkaline pH
(pH 7.9) of the culture environment in antibiofilm activity of ami-
noglycosides (e.g. gentamicin, tobramycin, and streptomycin) [44,60],
this condition was also applied in the present study. Before testing the
biofilm inhibition and virulence-attenuating properties against P. aer-
uginosa with these aminoglycosides, their MIC and sub-MIC values have
been determined using the alkaline pH (7.9). Results show that each
aminoglycoside exhibits different MIC values, which is in accord with
previous findings on tobramycin and streptomycin [44]. All the tested
aminoglycosides at their sub-MIC exhibited significant biofilm inhibi-
tion, although the inhibition was not dependent on concentration. This
effect is in accord with the biofilm inhibition properties of P. aeruginosa
by tobramycin and tetracycline in alkaline media, as recently reported
[44]. The biofilm inhibition properties by sub-MIC of aminoglycosides
were also visualized by SEM analysis, in which results demonstrate that
cells are less dense on the surface of the nylon membrane compared to
the control (Fig. 4). Interestingly, the morphology of the observed cells
was found to vary from rod-shaped to the filamentous, elongated, and
streptococcal forms. Kanamycin and netilmicin caused P. aeruginosa
biofilm cells to change to streptococcal cell shapes, whereas gentamicin
caused the bacterial cells to elongate at high sub-MIC levels (1 μg/ml)
and no changes at lower sub-MIC levels (0.25 μg/ml). Previous studies
have reported that the cell elongation process of P. aeruginosa occurs in
anaerobiosis and nitric oxide (NO) presence primarily to utilize the
limited oxygen source for proceeding cell growth [61–63]. The fila-
mentation and streptococcal development of the P. aeruginosa cell has
also been previously reported by Waisbren et al. [64] in the presence of
amikacin that is either treated singly or in combination with other
antibiotics. Thus, the present study suggests that the cell undergoes
morphology changes to cope with the effect of the sub-MIC of ami-
noglycosides; as such, due to morphology changes, biofilm inhibition is
not dependent on concentration (Fig. 3). A recent report showed that
the formation of the filamentous morphology of Vibrio cholerae cells
enables chitin surface attachment and dense biofilm architecture for-
mation [65]. Thus, the concentration-independent nature of biofilm
inhibition by these aminoglycosides may relate to the high amount of
dye that binds to the elongated and filamentous cells, as compared to
the rod and streptococci forms of the cells. Overall, this indicates that
F. Khan, et al. Microbial Pathogenesis 146 (2020) 104249

Fig. 6. Inhibitory effects of aminoglycosides at sub-MIC levels on the production of virulence factors of P. aeruginosa PAO1. (A) Pyocyanin production, (B)
Pyoverdine production and (C) LasA protease activity. The production of each virulence factor in the presence of aminoglycosides are represented a relative value
with respect to the control. All the experiments were performed in triplicates. *P < 0.05 and **P < 0.01 were accepted as statistically significant and ns indicated
non-significance.

Fig. 7. Inhibition of swarming motility of P. aeruginosa PAO1 by aminoglycosides at sub-MIC levels reflected by diameter value (cm) of swarming motility on agar
plates. A representative image of each assay is presented and each experiment was carried out three times. **P < 0.01 was considered as statistically significant.

7
F. Khan, et al. Microbial Pathogenesis 146 (2020) 104249

Fig. 8. Inhibition of swimming motility of P. aeruginosa PAO1 by aminoglycosides at sub-MIC levels reflected by diameter value (cm) of swimming motility on agar
plates. A representative image of each assay is presented and each experiment was carried out three times. **P < 0.01 was considered as statistically significant and
ns indicated non-significance.

Table 1
Interaction of different aminoglycosides with the LasR QS receptor of P. aeruginosa.
Target (LasR)
Ligand
Minimum binding energy (Kcal/ Hydrogen-bond(s) with Hydrophobic Interaction(s) with
mol)

3-Oxo-C12-HSL (OdDHL) −9.91 Tyr-56, Trp-60, Tyr-64, Asp-73, Arg-61, Ser- Tyr-47, Val-76, Cys-79, Leu-125
129
Gentamicin −12.25 Thr-75, Trp-60, Cys-79, Gly-126 Tyr-47, Gly-38, Tyr-64, Leu-40, Ala-50, Val-76, Gly-38, Leu-125,
Leu-36,
Tobramycin −10.47 Ser-129, Thr-75, Trp-60, Arg-61, Val-76 Gly-38, Leu-36, Ala-127, Tyr-64, Ala-50, Ile-52
Neomycin −4.66 Tyr-56, Trp-60, Tyr-64, Thr-115, Trp-88, Val-76, Leu-40, Ala-50, Leu-125, Ala-127
Ala-105
Kanamycin −10.21 Ser-129, Thr-75, Thr-115, Leu-125, Trp-60 Tyr-64, Leu-36,
Netilmicin −12.11 Ser-129, Thr-115, Thr-75 Tyr-64, Tyr-56, Trp-88, Leu-36
Paromomycin −6.36 Tyr-56, Trp-60, Tyr-64, Thr-115, Trp-88, Val-76, Ala-127, Leu-40, Ala-50
Tyr-93
Amikacin −15.24 Trp-60, Thr-75, Thr-115, Leu-40, Leu-39 Tyr-64,Leu-36, Ile-52,
Streptomycin −9.41 Tyr-47, Trp-60, Asp-65, Glu-48, Thr-75 Trp-88, Val-76, Leu-36, Ala-127, Ala-50, Tyr-64, Ser-129, Ile-52

the morphological changes of the bacterial biofilm cells can allow un-
derstanding the multi-drug resistance of the P. aeruginosa biofilm.
In addition to the various levels of inhibiting biofilm formation, the
sub-MIC levels of all tested antibiotics exerted eradication action to-
wards the pre-established mature P. aeruginosa biofilm. In the present
study, such action was most profound by gentamicin and neomycin.
The dispersal efficiency of the mature biofilm of P. aeruginosa by these
aminoglycosides was found to be similar to those of streptomycin and
tobramycin, as reported recently [44]. Furthermore, several previous
studies have reported several modifications of these antibiotics, such as
featuring conjugation onto nanocarriers or combination with other
drugs, have successfully eradicated the mature biofilm [66,67]. In the
present study, the modified pH of the culture environment may have
facilitated drug-eradicating activity. As P. aeruginosa is a nosocomial
pathogen that colonizes on several types of surfaces (e.g. medical de-
vices and food processing equipment) to cause infections, an antibiotic
effectively exhibiting both biofilm inhibition and pre-existing mature
biofilm eradication against the bacteria is considered highly potential
[68–70].
For the present study, the sub-MIC levels of several aminoglycoside
drugs were evaluated for their actions against P. aeruginosa virulence
factors, which included QS-regulated pyocyanin, pyoverdine, and LasA
protease enzymes. In P. aeruginosa, pyocyanin plays a crucial role in
causing cystic fibrosis and lung infections [71–73]; whereas pyoverdine

8
F. Khan, et al. Microbial Pathogenesis 146 (2020) 104249

Fig. 9. Aminoglycosides docked in different binding pockets of LasR of P. aeruginosa. (A) 2D interaction of amikacin, gentamicin, kanamycin, neomycin and
netilmicin with the LasR active site, (B) 3D interaction of amikacin, gentamicin, kanamycin, neomycin and netilmicin with the LasR active site, (C) 2D interaction of
paromomycin, streptomycin, tobramycin and N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL) with the LasR active site and (D) 3D interaction of paromomycin,
streptomycin, tobramycin and N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL) with the LasR active site.

is produced for bacterial metabolism in an iron-limited environment
[46,74,75]. Protease enzymes, such as protease A (LasA), elastase B
(LasB), and alkaline enzyme, are crucially utilized for bacterial colo-
nization and host cell damage [76–78]. Overall, results have shown that
the inhibitory action of sub-MIC levels of tested antibiotics was most
effective against pyoverdine production, pyocyanin production, and
LasA protease activity of P. aeruginosa.
Motility is also considered among the virulence properties of P.
aeruginosa [79]. The surface movements, such as swimming and
swarming by flagella, play a determining role in the bacterial initial
attachment and colonization processes [79–81]. In the present study,
the treatments of various antibiotics were found to cause significantly
different inhibitory effects on P. aeruginosa motility. For both the
swimming and swarming movements, the high sub-MIC levels of all
tested antibiotics were more effective than the lower ones.
It is well noted that biofilm establishment and virulence properties
are majorly regulated by the following two QS cell-to-cell commu-
nication systems in P. aeruginosa: the las and rhl systems [82–84]. The
former system (i.e. las system), via interactions between the transcrip-
tional regulator LasR and 3O–C12-HSL diffusible signal molecules that
are encoded by lasI cognate acyl homoserine lactone synthase, is re-
sponsible for the activation of lasA, lasB, and lasI, which encode for
various protease enzymes, such as LasA protease, LasB elastase, and
alkaline protease [85,86]. As aforementioned, these enzymes play an
important role in P. aeruginosa pathogenesis [87–89]. In addition to
protease enzymes, previous studies demonstrate that LasR is also in-
volved in regulating the rhl system, biofilm formation, and the pro-
duction of other virulence factors (e.g. pyocyanin and rhamnolipid)
[90,91]. Thus, in the present study, we have further performed in silico
docking analysis to evaluate molecular interactions between QS-asso-
ciated LasR proteins and different aminoglycosides. Docking results
indicate that the aminoglycosides with inhibition against pyocyanin
and LasA protease also exhibit high affinity binding to the active site of
LasR. The mode of this binding was found to involve hydrogen bonds
and hydrophobic interactions, which is similar to several antibiofilm
agents that were previously reported (e.g. vitexin and 6-gingerol)
[24,90]. Overall, it can be inferred that the antibiofilm activities of
these antibiotics are derived from targeting the QS regulatory protein
LasR. Thus, based on the present findings, modifying the culture en-
vironment and concentration optimization results in the revitalization
of antibiotics as an antibiofilm and antivirulence agent to treat biofilm-
forming pathogenic bacteria.
5. Conclusion
In the present study we selected several aminoglycosides and per-
formed biofilm inhibition related assays using their sub-MIC levels and
alkaline (pH 7.9) culture environment. Each aminoglycoside was found
to exhibit different inhibition level towards P. aeruginosa biofilm for-
mation, as well as different eradicating activity to the bacterial pre-

9
F. Khan, et al.
established mature biofilm. The sub-MICs of aminoglycosides also
showed anti-virulence activity against the bacterial motility, as well as
the production of pyocyanin, pyoverdine and LasA protease enzymes,
which is likely to derive from their binding with QS regulatory mole-
cule. For future studies, it is suggested that the effect of the tested an-
tibiotics on other virulence factors should be exploited. In addition,
molecular studies on the drug binding with different QS associated
proteins are required to further confirm the mechanism of antibiofilm
activities of these aminoglycosides at sub-MIC level.
Compliance with ethical standards
This article does not contain any studies with human participants or
animals.
Funding information
This work was supported by Basic Science Research Program
through the National Research Foundation of Korea funded by the
Ministry of Education (NRF-2019R1A2C1087156).
Author statement
Fazlurrahman Khan: Conceptualization, Investigation,
Methodology, Validation, Writing and Editing.
Jang-Won Lee: Methodology.
Aqib Javaid: Methodology, Validation and Writing.
Seul-Ki Park: Methodology.
Young-Mog Kim: Conceptualization, Funding acquisition, Editing
and Supervision.
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgement
Author would like to thank Dung Thuy Nguyen Pham for her
technical help during the experiment and language editing.
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