Microbial Pathogenesis 149 (2020) 104480

Contents lists available at ScienceDirect

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

Evaluation of behavior, growth, and swarming formation of Escherichia coli

and Staphylococcus aureus in culture medium modified with
silver nanoparticles
Lucio Assis Araujo Neto a, b, Tatiane Melo Pereira b, c, Luciano Paulino Silva a, b, c, *
a
Embrapa Genetic Resources and Biotechnology, Laboratory of Nanobiotechnology (LNANO), Brasilia, 70770-917, DF, Brazil
b
Federal University of Parana (UFPR), Postgraduate Program in Pharmaceutical Sciences, Curitiba, 80210-170, PR, Brazil
c
University of Brasilia (UnB), Postgraduate Program in Nanoscience and Nanobiotechnology, Brasilia, 70910-900, DF, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Silver nanoparticles (AgNPs), as well as silver ions, are described as toxic to a broad spectrum of microorganisms,
Silver nanoparticles especially bacteria. In contrast to this, a current trend is to develop and carry out the in vitro cultivation of
Green synthesis microorganisms, facilitating the study of interactions between populations of cells and species. Thus, the goal of
Swarming
this study was to evaluate the behavior, growth, and swarming formation of bacteria under conditions of co-
Toxicity
Modified culture medium
culture in solid medium modified with AgNPs. The aqueous extract from the leaves of Handroanthus serratifo­
lius was used to chemically reduce silver nitrate (AgNO3) solution, forming AgNPs. This synthesis route was
performed in an aqueous medium at 50 ◦ C for 3 h. The hydrodynamic diameter (HD) and polydispersity index
(PdI) were obtained by dynamic light scattering (DLS), and Zeta potential (ZP) of the AgNPs were measured by
electrophoretic mobility. Atomic force microscopy (AFM) was used to evaluate the shape of the AgNPs. Luria
Bertani (LB) medium was used for the liquid culture steps and for the solid medium, bacterial agar was added.
Solutions containing AgNPs or AgNO3 were added at final concentrations of 256, 128, or 64 μM. Subsequently,
microorganism Escherichia coli ATCC® 8739 and Staphylococcus aureus ATCC® 25923 were plated with AgNPs,
AgNO3, and control media. Analyses of the AgNPs showed an average HD of 76.02 ± 3.08 nm, PdI of 0.461 ±
0.012, and ZP of − 21.5 ± 2.2 mV; in addition, AgNPs were nearly spherical. The solid culture medium elaborated
and modified with AgNPs at the concentrations of 256 and 128 μM inhibited the growth of the tested micro­
organisms and decreased the swarming formation. However, those media modified at a concentration of 64 μM
did not induce any alteration in the growth and proliferation of the microorganisms. Furthermore, it was
observed that plates containing modified culture media with 128 μM, increased proximity between both co-
cultured bacteria occurred. Thus, the application of AgNPs in solid culture media becomes a promising and
potentially reproducible strategy for evaluating the behavior, swarming formation, and toxicity of AgNPs,
making the understanding of possible bactericidal or bacteriostatic effects, and also colonizing strategies.

1. Introduction approaches based on the synthesis of this nanomaterial by green routes

Silver nanoparticles (AgNPs), as well as silver ions (Ag+), are
described as promising materials in medical and biotechnological ap­
plications due to their high antimicrobial efficiency, particularly against
bacteria [1,2]. However, when in the form of nanostructures, there is an
increase in the surface area for the exposure of this material to a
microorganism potentially increasing the inhibitory effect [3] and also
presenting minimal or no cytotoxicity towards human cells [4]. As an
inexpensive alternative with an eco-friendly appeal emerges those
using aqueous plant extracts [5,6] or microorganisms [7] for the
reduction of AgNO3 and consequently the formation and stabilization of
AgNPs.
Among the assays for the identification of antimicrobial activity are
the minimum inhibitory concentration (MIC) [8], the inhibition halo
assay [9], and the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazo­
lium bromide (MTT) [10]. These, in turn, are typically biological tests
for the detection and evaluation of nanomaterial toxicity [11]. On the
other hand, when microorganisms participate in a series of survival

* Corresponding author. Embrapa Recursos Genéticos e Biotecnologia (Cenargen), Parque Estação Biológica, Final W5 Norte, Asa Norte, 70770-917, DF, Brazil.
E-mail addresses: luciano.paulino@embrapa.br, lucianopaulinosilva@gmail.com (L.P. Silva).

https://doi.org/10.1016/j.micpath.2020.104480
Received 3 September 2019; Received in revised form 10 February 2020; Accepted 28 August 2020
Available online 12 September 2020
0882-4010/© 2020 Elsevier Ltd. All rights reserved.
L.A. Araujo Neto et al. Microbial Pathogenesis 149 (2020) 104480

strategies (in response to dangerous substances or in a competition),

they present unprecedented evading mechanisms. During the cultiva­
tion of bacteria in solid media, the swarming process occurs as a
mechanism to search other sources of nutrients, increase their size, and
colonize niches [12]. Thus, a current challenge regarding the growth of
microorganisms is to carry out the in vitro co-culture approach since it
allows the study of their interactions under normal or altered conditions
[13].
Therefore, the aim of the present study was to evaluate the behavior,
growth, and swarming formation of bacterial colonies of Escherichia coli
and Staphylococcus aureus submitted to co-cultivation in a solid medium,
with or without AgNPs or AgNO3 addition in different concentrations.

2. Material and methods

2.1. Synthesis of silver nanoparticles (AgNPs)

Botanical material (leaves) of Handroanthus serratifolius (A. H.

Gentry) S. Grose was collected at Botanical Garden of Brasília (JBB)
under license numbers (SISBIO:57671-1; JBB:008/2017;
CGEN:02001007580/2014-95), and stored frozen at − 20 ◦ C until the
moment of use. Leaves were washed with Extran® 0.1% for 2 min. Thus,
these leaves have been cut in fragments of about 5 mm2 and boiling
water was added up to a concentration of 100 mg/mL was reached and
the resulting aqueous extract was further filtered. Aqueous leaves
extract of H. serratifolius at the final concentration of 1 mg/mL was used
as a bio-reducing agent of the 1 mM AgNO3 solution. Silver ions
reduction reaction was performed in a water bath at 50 ◦ C for 3 h.
Monitoring of AgNPs formation was performed visually through the
color change of the suspension monitored at 450 nm every 30 min using
a UV–Vis spectrophotometer (Quimis, Brazil). Controls of AgNO3 solu­
tion and of extract were prepared following the same procedure
described above.

2.2. Characterization of AgNPs

Samples were diluted 1:10, UV–Vis analyses were performed

following analyses by dynamic light scattering (DLS) and Zeta potential
(ZP). In UV–Vis, the absorption spectra were scanned over the 350–550
nm wavelength range on a spectrophotometer using a 1 cm path length
plastic cuvettes. DLS and ZP were both measured on the equipment
ZetaSizer Nano ZS (Malvern, UK). DLS was performed with an angle of
173◦ using He–Ne laser (4 mW) operating at 633 nm and three mea­
surements were obtained at 25 ◦ C in automatic running mode. ZP also
used the same number of replicates and temperature with the running of
twenty acquisitions.
2.3. Atomic force microscopy
The shape of the AgNPs (height) were evaluated by atomic force
microscopy (AFM), using the SPM-9600 (Shimadzu, Japan) equipment
operated at ambient temperature (~22 ◦ C). Approximately 2 μL of the
diluted sample (1:10) were deposited on a freshly-cleaved muscovite
mica surface and allowed to dry. Subsequently, the images were ac­
quired in dynamic-phase mode, scanning in XY directions (10 μm × 10
μm) at a scan rate of 1 Hz, using conical silicon tips integrated into
rectangular cantilevers with a resonance frequency of about 250–300
kHz. Finally, the offline software SPM-9600 was used to correct the
planes and inclinations of the X-axis and background on both axes.
Fig. 1. UV–Vis spectrophotometric assessments of AgNPs. a) Evaluation of the
formation of AgNPs at 450 nm every 30 min for 3 h. b) Evaluation of the
maximum absorption peak between 350 and 550 nm of the AgNPs, with a 10
times dilution.
(Sigma L3022) in 50 mL polypropylene tubes and incubated at 37 ◦ C
overnight in a horizontal shaker (LUCA-222) at 120 rpm. By using a
Biophotometer (Eppendorf), optical density at 600 nm (OD600) of the
bacteria was obtained and later diluted to the final value of 0.05 A U.
(Arbitrary Units) in each mL.
2.5. Preparation of the culture medium
The solid culture media were prepared in flasks using 2% w/v of LB
medium and 1.5% w/v bacteriological agar (Vetec). After wet heat
sterilization, they were poured into sterile polystyrene Petri dishes of 10
cm diameter (JProlab) and after solidification of the medium at room
temperature were stored at 4 ◦ C until use.
2.6. Preparation of the modified culture media

2.4. Cell culture
For the bacteria growth assays, strains belonging to two bacteria
species were used, the Gram-positive S. aureus ATCC® 25923 and the
Gram-negative E. coli ATCC® 8739. These were collected from indi­
vidually cultured colonies using 2% w/v Luria Bertani (LB) medium
Modified solid culture media were prepared in Erlenmeyer flasks,
using 20 mg/mL of LB medium and 1.5% bacteriological agar. After wet
sterilization using autoclave process, the AgNPs and AgNO3 solution
were added to the respective flasks followed by manual shaking to ho­
mogenize the solution to reach the final concentrations of 256, 128, or
64 μM. Then, the modified media were poured into 10 cm diameter

2
L.A. Araujo Neto et al. Microbial Pathogenesis 149 (2020) 104480

Fig. 2. Distribution of the hydrodynamic diameter in number (%) of the AgNPs

produced with the aqueous extract of Handroanthus serratifolius.

polystyrene Petri dishes which were stored at 4 ◦ C after solidifying at

room temperature.

2.7. Swarming assay

Microorganisms were added to the plates with modified bacterial

culture media, forming distinct designs during pipetting. On the first
design, E. coli was spotted to the middle; and in another design, S. aureus
was spotted in the middle, both for the evaluation of the possible
swarming process of these bacteria. At the third design, random drops of
E. coli were deposited onto the plate and on the fourth design each
microorganism strain was added to the central portion thereof. All the
plates were maintained in the bacterial incubator (LUCA-81/81) at
37 ◦ C, evaluated visually every day, monitored the growth by recording
photos, and evaluated swarming formation in alternate days.

2.8. Toxicity test towards bacteria exposed to modified culture media

Microorganisms were added to the plates with modified bacterial

culture media, forming distinct designs during pipetting. In 6 plates, one
drop of E. coli was added to the medium and then drops of S. aureus were
deposited around them. In the other 6 plates, the opposite was done, that
means, one drop of S. aureus was added to the medium and then drops of
E. coli were deposited around them. Plates were maintained in the
bacterial incubator at 37 ◦ C for 14 days and visually evaluated daily,
recording the growth and swarming formation of each species on the
control plates and also those with modified media.
3. Results and discussion
The progress of the formation of AgNPs was monitored visually
during reaction synthesis. The color of the suspension changed from
light yellow to light brown. The formation curve (Fig. 1a) demonstrated
that there was an increase in absorbance as a function of time, corrob­
orating the formation of AgNPs. In addition, the characterization by the
UV–Vis spectrophotometry (Fig. 1b) showed a maximum absorption
peak at 440 nm, within the expected range of 400–450 nm typical
indicative of the presence of AgNPs [14]. Characterization methods such
as DLS and ZP were applied to measure the hydrodynamic diameter and
surface Zeta potential of the formed AgNPs, respectively. DLS analysis
indicated that the mean hydrodynamic diameter (Z-average) of the
AgNPs was 76.02 ± 3.08 nm, and in Fig. 2 their size distribution (%) in
number is shown. It is noteworthy that these values are very different
due to distinct ways used to calculate both size indicators for relatively
Fig. 3. Atomic force microscopy images of AgNPs synthesized by aqueous ex­
tracts of Handroanthus serratifolius. The topographic images of height corre­
spond to the acquisition in the dynamic-phase mode, with a resolution of 512 ×
512 lines and a scan rate of 1 Hz. a) Top-view image of AgNPs. b) Three-
dimensional view of AgNPs.
polydisperse suspensions and in this case the presence of molecules of
plant extract could also contribute to such differences. In fact, PdI value
identified was 0.461 ± 0.012, indicating moderate particle poly­
dispersity. Particles around this size and polydispersity have already
shown that can interact with bacteria, causing their death [15,16]. The
ZP demonstrated incipient colloidal instability with a value of − 21.5 ±
2.2 mV [17].
The images acquired from the AgNPs by AFM reveal that they have a
near spherical shape and that in some structures it was possible to
observe the coating formed by metabolites of the extract, which are
responsible for the stabilization of the nanoparticles (Fig. 3a for top-
view). Meanwhile, Fig. 3b shows three-dimensional representation of
the mica surface deposited with the AgNPs and it was observed that the
maximum height (dry-diameter) of the AgNPs (~58.8 nm) was below
the value of the Z-average. As expected, AFM demonstrated a lower size
of the nanoparticles when compared to their HD measured by DLS. This
happens because in this case the material is in a dry environment and in

3
L.A. Araujo Neto et al. Microbial Pathogenesis 149 (2020) 104480

Fig. 4. Growth and swarming formation of bacteria after 14 and 8 days of incubation. The group of images represented in 4a represents the evolution of the natural
swarming of E. coli and in 4b, we observed the formation of natural swarming for S. aureus, evaluated in fourteen days. In the image represented by 4c, is found the
growth of four colonies of E. coli dripped separately showing their behavior with the same microorganism sample, and at 4d the co-cultivation of both bacteria and
the swarming formation is visualized, noting that the swarming of S. aureus (red arrow) is inhibited by E. coli (yellow arrow) after 8 days.

HD the nanoparticles are in solution. Then it is estimated that it is
possible to expect a difference of up 30–40% of the diameter when
comparing the same material by both techniques [18].
During the growth of bacteria, each culture plate was photographed
to follow the swarming formation for each microorganism. Fig. 4a and b
represent the evolution of the natural swarming of E. coli and S. aureus,
respectively, evaluated after fourteen days. Fig. 4c shows the growth of
four colonies of E. coli dripped separately showing their behavior with
the same microorganism sample. Otherwise, Fig. 4d shows the co-
cultivation of both bacteria and the swarming formation is visualized,
noting that the swarming of S. aureus is inhibited by E. coli after 8 days
indicating the association incompatibility between them. The presented
models of growth are similar and are in agreement with those described
by the other researchers, mainly to the studied microorganisms [19,20].
After maintenance of the microorganisms in plates with modified
culture medium, their behaviors were analyzed during 14 days to un­
derstand the possible influence of an environment with good nutritional
conditions of growth, but with factors that could prevent their pro­
liferations including antibacterial nanoparticles or other microorganism
colonies. Fig. 5a shows the co-cultivation of the microorganisms at the
14th day of observation inoculated in LB medium without modification.
On both occasions, the bacteria placed in the center of the plate have
restriction to growing. However, those deposited around, have higher
performance and swarming formation. The physical non-interaction
between them can be explained by metabolites exuded by themselves
and that have the antimicrobial capacity so that organisms that are not
of the same species can interact [21,22].
Fig. 5b, d, and f show the bacteria added in AgNPs-modified medium
at the concentrations of 256, 128, and 64 μM, respectively. How higher
the concentration of AgNPs implied in inhibition of growth of the mi­
croorganisms of both strains, how higher the cytotoxic activity. At 128
μM, E. coli had a much lower growth and development rate than
S. aureus, which is explained by differences in the composition of their
cell walls, which differ between Gram-positive (more thickness) and
Gram-negative (less thickness) in that permeability of AgNPs is favored.
In contrast, when exposed to the lowest concentration, both bacteria
grew normally, except for those that were deposited onto the center of
the plates.
Fig. 5c, e, and g show bacteria spotted onto AgNO3-modified culture
media at concentrations of 256, 128, and 64 μM, respectively. S. aureus
and E. coli showed distinct behavior after contact with each modified
culture medium during the fourteen days. As in the plates containing
AgNPs in higher concentrations, the AgNO3 presented equivalent cyto­
toxic activity, inhibiting the growth of both bacteria strains. Differently,
at the concentration of 128 μM, S. aureus was able to develop well in
comparison to E. coli, which did not grow as well, except when evaluated
in the plate shown in Fig. 5e, where a colony probably became resistant
to silver or even by the irregular homogenization of the nanomaterial in
the medium allowing the bacteria to proliferate, even in the presence of
another microorganisms of different species. At the lower concentration

4
L.A. Araujo Neto et al.
of AgNO3, it was observed that the growth, development, and formation
of swarming remained similar to the plates containing the medium
modified with 64 μM AgNPs and the plate with culture medium without
change.
The solid culture media modified with the AgNPs suspension or
AgNO3 solution in the three concentrations were able to inhibit or not to
hinder the growth and swarming formation of the microorganisms when
compared to the control plates. Co-cultivation of microorganisms in the
solid medium is not yet well established, this being more common in a
liquid culture medium. Furthermore, there is a report of co-cultivation
using E. coli, Bacillus subtilis, and Shewanella oneidensis for the produc­
tion of chemicals as secondary metabolites [23]. Those authors
demonstrated that this type of technique possessed significant produc­
tion advantages over similar monoculture strategies. In contrast, a
semi-permeable co-cultured system containing E. coli (K12), Pseudo­
monas aeruginosa (PA14), and Burkholderia cenocepacia (K56-2) sepa­
rated by membrane barriers was used to evaluate and quantify
independent interactions among bacterial species/populations, for the
diffusion of molecules [24]. Moreover, the relationship between
S. aureus and P. aeruginosa in co-culture on a bronchial epithelial
monolayer (CFBE) was studied in order to evaluate its possible influence
on cystic fibrosis [25].
The research and studies of the behavior of microorganisms for
resistance to diverse types of antibiotics is remarkable the variation of
defenses that have been developed over the decades. For E. coli, the
resistance responses found in the literature are for ampicillin, cephalo­
sporins, chloramphenicol, fluoroquinolones, nalidixic acid rifampin,
5
Microbial Pathogenesis 149 (2020) 104480
Fig. 5. Growth and formation of bacterial
swarming in normal medium andmediamodified
with AgNPs and AgNO3 evaluated for 14 days. In
the two plates indicated by 5a are the bacteria
added in LB medium, without modification. The
group of images shown in 5b, 5d, and 5f shows
the bacteria added in AgNPs-modified medium at
the concentrations of 256, 128, and 64 μM of
silver, respectively. In the group of images rep­
resented by 5c, 5e, and 5g, the bacteria were
added in the culture medium modified with sil­
ver nitrate in the concentrations of 256, 128, and
64 μM, respectively. S. aureus (red arrow) and
E. coli (yellow arrow) after 14 days showed
distinct behavior after contact with modified
culture media. Media containing 256 and 128 μM
of AgNPs or AgNO3 altered the swarming for­
mation of both microorganisms.
and sulfamethoxazole streptomycin tetracycline. To S. aureus is already
found for methicillin and vancomycin [1]. This allows for the develop­
ment of experimental strategic models which aim to obtain alternative
forms of microbiological control using new antimicrobial technologies.
The use of nanomaterials, especially AgNPs, has become an
increasingly sought after option for antimicrobial activity [26]. How­
ever, there are discussions about the resistance of microorganisms to
nanoparticles [27]. Thus, this work demonstrates the application of
AgNPs and silver solution acting as a bactericidal agent for E. coli and
S. aureus in higher concentration when added to the solid culture me­
dium, inhibiting or at least modulating the formation of swarming.
However, when placed in modified culture media in a lower concen­
tration, there is a good swarming development, including the possible
co-cultivation of both microorganisms.
4. Conclusion
Solid culture media that are suitable for microorganisms propel them
to proliferate actively and steadily in order to access new sources of
nutrients, increase community size, and colonize different niches. In
contrast, when these environments are modified, there is a change in
their behavior. The present study showed that when AgNPs or silver
solutions are added to the solid culture medium of bacteria, in moderate
concentrations, they are able to inhibit the growth, development, and
formation of swarming. At lower concentrations, there are no behavioral
changes. Our study was able to correlate the addition of a silver-based
nanomaterial with antimicrobial activity in solid culture medium and
L.A. Araujo Neto et al.
modulate the growth during co-cultivation of two bacterial strains. We
have found that AgNPs at the concentration of 256 μM can inhibit the
growth of both microorganisms and 128 μM can impair the growth of
E. coli. Therefore, we understand that this approach is innovative and
potentially reproducible for the analysis of the behavior and formation
of swarms for other species of microorganisms, as well as to evaluate
possible mechanisms of resistance to antibiotics as well as the toxicity of
nanomaterials.
CRediT authorship contribution statement
Lucio Assis Araujo Neto: Conceptualization, Methodology, Vali­
dation, Writing - original draft. Tatiane Melo Pereira: Conceptualiza­
tion, Methodology, Validation, Writing - original draft. Luciano
Paulino Silva: Conceptualization, Writing - review & editing, Supervi­
sion, Funding acquisition.
Declaration of competing interest
The authors declare that there is no conflict of interests regarding the
publication of this paper and the funding agencies have not competing
interests with the conduct of the research and/or preparation of the
manuscript.
Acknowledgments
We appreciate the financial support of the Brazilian agencies Coor­
denação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES
Finance Code 001 and 23038.019088/2009-58), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq no. 307853/2018-7,
408857/2016-1, 306413/2014-0, and 563802/2010-3), Fundação de
Apoio à Pesquisa do Distrito Federal (FAPDF no. 193.001.392/2016),
Empresa Brasileira de Pesquisa Agropecuária (Embrapa no.
23.17.00.069.00.02, 13.17.00.037.00.00, 21.14.03.001.03.05,
13.14.03.010.00.02, 12.16.04.010.00.06, 22.16.05.016.00.04, and
11.13.06.001.06.03), Universidade de Brasília, Universidade Federal do
Paraná, and help of all members of the Laboratory of Nano­
biotechnology (LNANO) of Embrapa.
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