Methods Article

Progress in Microbes and Molecular Biology

An Optimized Anti-adherence and Anti-biofilm Assay: Case Study of

Zinc Oxide Nanoparticles versus MRSA Biofilm
Hefa Mangzira Kemung1,2, Loh Teng-Hern Tan2, Kooi Yeong Khaw1, Yong Sze Ong1,3, Chim Kei Chan4,
Darren Yi Sern Low5, Siah Ying Tang5,6, Bey-Hing Goh1,6,7*
1
Biofunctional Molecule Exploratory Research Group (BMEX), School of Pharmacy, Monash University Malaysia, 47500
Bandar Sunway, Selangor Darul Ehsan, Malaysia
2
Novel Bacteria and Drug Discovery Research Group (NBDD), Microbiome and Bioresource Research Strength, Jeffrey
Cheah School of Medicine and Health Science, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan,
Malaysia
3
Health and Well-being Cluster, Global Asia in the 21st Century (GA21) Platform, Monash University Malaysia, Bandar
Sunway 47500, Malaysia
4
de Duve Institute, Avenue Hippocrate 75, 1200 Brussels, Belgium
5
Chemical Engineering Discipline, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500
Bandar Sunway, Selangor, Malaysia
6
Advanced Engineering Platform, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor,
Malaysia
7
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

Abstract: Biofilms form protective layers over bacteria that are associated with a majority of the hospital infections
contributing to antibiotic resistance development in susceptible strains. Nowadays, there is a pressing need for developing
effective anti-biofilm agents to help address the growing problem of biofilm-producing bacteria associated with antibiotic
resistance. In recent years, zinc oxide nanoparticles (ZnO-NPs) has emerged as a prospective candidate for new anti-biofilm
agents. The present method paper described an optimized anti-adherence and anti-biofilm assay using ZnO-NPs. The
antibiotic-resistant bacteria Methicillin-resistant Staphylococcus aureus (MRSA ATCC4330) and vancomycin were used as
the growth control and positive control, respectively. The result showed concentration-dependent anti-adherence and anti-
biofilm activity. The ZnO-NPs effectively prevented attachment of bacterial cells onto walls of wells with 51.69 ± 2.55% at
the highest concentration tested (65.4 µg/mL). ZnO-NPs was also able to break-up 50% pre-formed MRSA biofilm at the
lowest concentration of 13.5 µg/mL. Interestingly, ZnO-NPs at lower concentrations demonstrated significantly stronger anti-
biofilm activity than that of the positive control vancomycin, demonstrating that ZnO-NPs is a promising anti-biofilm agent.
This method could be used as a preliminary screening of transition metal oxide nanoparticles as potential anti-adherence and
anti-biofilm agents followed by other specific anti-biofilm assays.

Keywords: Methicillin-resistant Staphylococcus aureus; MRSA; anti-biofilm; anti-adherence; Zinc oxide nanoparticles;
ZnO-NPs

*Correspondence: Bey-Hing Goh, School of Pharmacy, Monash

Received: 3 May 2020
rd University Malaysia, 47500, Bandar Sunway, Selangor Darul
Accepted: 3rd June 2020 Ehsan, Malaysia; [email protected]
Published Online: 12th June 2020

Citation: Kemung HM, Tan LT-H, Khaw KY, et al. An Optimized Anti-adherence and Anti-biofilm Assay: Case Study of
Zinc Oxide Nanoparticles versus MRSA Biofilm. Prog Microbes Mol Biol 2020; 3(1): a0000091. https://doi.org/10.3687/
pmmb.a0000091.

Introduction activities[1–18]. Even to this day, nature continues to

instill its significance in society as a prominent resource
Over the course of history, nature through its subsidiary for future antibiotics in treating antibiotic-resistant
plants and microbes, has proven to be an essential player infections[19]. Despite the use of current antibiotics,
in driving development of future drugs by virtue of their infectious diseases acquired either in hospitals or through
potential in producing secondary metabolites with anti- consumption of foods contaminated by food-borne
bacterial, anti-cancer, anti-oxidant and neuroprotective

Copyright @ 2020 by Kemung HM and HH Publisher. This work under licensed under the Creative Commons Attribution-NonCommer-
cial 4.0 International Lisence (CC-BY-NC4.0)
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 An Optimized Anti-adherence...

pathogens[20–26] remain a major public health problem. (MRSA) ATCC 43300 and vancomycin hydrochloride
Additionally, the unscrupulous practice of antibiotics for were used as the control bacteria and positive control,
various ailments has encouraged antibiotic-susceptible respectively. The experiment set-up consisted of a 96-
infectious bacteria to form natural defenses against well plate with a flat bottom, crystal violet as a staining
them. One such defense established by these bacteria is agent and a 96-well microplate reader for quantification
the biofilm[19]. of both the anti-adherence and anti-biofilm activities. The
result obtained indicate ZnO-NPs has anti-adherence and
Biofilm is a term that describes a community of anti-biofilm properties against MRSA ATCC 4330. Given
microorganisms within a self-produced matrix of that crystal violet anti-adherence and anti-biofilm assay is
biopolymers attached on surfaces[27]. Microbes tend to an indirect measure of biofilm biomass, this study could
produce biofilm on surfaces evading harmful effects of be used as a preliminary screening to investigate the anti-
antibiotics as well as detergents and persists in hospitals biofilm properties of transition metal oxide nanoparticles
causing many internalized hospital-related infections. It prior to studying the mechanism of action of anti-adherence
was estimated that biofilm contributes to approximately and anti-biofilm properties.
60 to 80% of hospital infections[28–30]. Given that
Staphylococcus aureus normal flora is the skin, suggests Method Details
that it is among the most common causative agent in
hospital-acquired infections associated with medical Synthesis of ZnO-NPs
implants[31,32]. Moreover, it was shown that S. aureus
was tolerant against higher doses of antibiotics and may A weighted measurement of 1.90 g of zinc nitrate
thus contribute to development of antibiotic resistance in hexahydrate (Zn(NO3)2.6H2O) is first dissolved in 100 mL
susceptible strains[32]. of ultrapure water under constant stirring. Subsequently,
the pH of the mixture was adjusted to pH 10 using 1M of
Recent years has seen a growing interest in the study of sodium hydroxide (NaOH) solution. Next, the solution is
biofilm inhibitors acting as adjuvant agents in reducing heated for 1 hour at 85°C under continuous stirring. The
biofilm layer of pathogenic bacteria[33]. This has led to the white suspension was then centrifuged for 5 minutes at
use of anti-biofilm assays to identify alternative sources 7000 rpm. Upon removing the supernatant, the residue is
as potential inhibitors of microbial biofilm. Previous washed with distilled water and then subjected to another
studies have highlighted the antibacterial potential of cycle of centrifugation before removing the supernatant
transition metal oxides for crop protection[34] and disease again. The residue was then dried in an aerated oven at
eradication[35–37]. Nanoparticles especially those of 60°C overnight, yielding a white powder.
metallic nature are one of the newest emerging systems
which have great potential in inhibiting the formation Anti-adherence and Anti-biofilm Assay
of biofilms accredited to their high anti-microbial and
anti-bacterial properties. The use of nanoparticles as
Materials
anti-biofilm agents have found its way in many different • Biosafety level 2 cabinet, functional incubator,
sectors such as in healthcare (drug delivery, therapeutics 96-wells microplate reader
and dentistry) or even in the food industry with a plethora
of tailored applications[38,39]. • Sterile disposable consumables: 96-wells microtiter,
15 mL centrifuge tubes
The potency of these metallic nanoparticles in resisting
the production of biofilm is high due to its nanoscale size • Bacterial cells (American type culture collection
and active participation in most of the stages in biofilm strain- ATCC)
production. If the nanoparticles can successfully prevent
• Bacteria nutrient-rich media. The use of Tryptic soy
adherence of microbes, then cycle of biofilm production
broth (TSB) which contains glucose and stimulates
is halted from the start. Sometimes, these nanoparticles
biofilm formation especially with Methicillin-resistant
disrupt the biofilm at the proliferation or even maturation
Staphylococcus aureus.
stages, generally through the formation of radicals and
reactive oxygen species (ROS) which affects gene • Aqueous crystal violet (0.1% w/v)
expressions and breaks DNA strands[40]. In this context,
the ZnO-NPs were chemically synthesized using a zinc • Glacial acetic acid (30% v/v)
nitrate precursor and subsequently characterized to
• 1×Phosphate buffer saline
confirm its identity. This includes conducting elemental
analysis, Fourier-transform infrared spectroscopy and • Multichannel pipette (preferable)
morphological analysis using electron microscopy. The
ZnO-NPs synthesized as an anti-adherence and anti- • Vancomycin hydrochloride drug as the positive control
biofilm agent have nanorice morphologies and have an
• Zinc oxide nanoparticles (ZnO-NPs) prepared in
average size of 250 nm.
different test concentrations
The aim of this methodology article is to present step-by-
step and optimized anti-adherence and anti-biofilm assays Procedure
to evaluate the efficacy of ZnO-NPs as anti-adherence
Bacterial culture preparation
and anti-biofilm agents[41] (Figure 1). To validate the test
method, Methicillin-resistant Staphylococcus aureus Inoculate 3 to 5 pure colonies of MRSA ATCC43300 from

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 Kemung HM et al.

the culture plate into 15 mL TSB. Revive the bacteria 600 nm)
in the shaker incubator at 200 rpm and at 37°C for
18 to 24 hours prior to the experiment so that they 11. Calculate the anti-adherence activity of test substance
are preferably in their log phase of growth. Ensure and vancomycin using the following formula:
sterile TSB is used by autoclaving TSB at 121°C for
15 minutes. Absorbance of control-Absorbance of test sample
Anti-adherence activity% = × 100%
Absorbance of control
Anti-adherence Assay

1. Inoculate 50 μL of ZnO-NPs and vancomycin Anti-biofilm Assay

into designated wells at a series of concentration.
(vehicle control e.g. DMSO is also needed to be 1. Follow the steps stated in bacterial culture preparation
aliquoted into appropriate wells if used as the and step 2 in anti-adherence assay to prepare 106 CFU/
diluent for the test substance) mL bacterial suspension. Inoculate 100 μL of the diluted
bacterial suspension in TSB into respective well of a
2. Prepare a bacterial suspension (~1 x 108 CFU/mL
new 96 well microplate.
equivalent to UV absorbance reading of 0.08 to
0.1 with wavelength at 600 nm) in 15 mL from 2. Incubate the plate at 37oC in an incubator for 24 hours.
a 24-hour bacterial culture. Alternatively, a 0.5
McFarland standard can be used to determine 3. Decant the TSB broth completely from the microplate,
the optimal bacterial suspension. Make a 1:100 wash the well gently without disrupting the biomass
dilution in a separate centrifuge tube to obtain a formed attaching on the bottom and wall of the wells
106 CFU/mL bacterial suspension. with sterile phosphate buffer saline (PBS) 3 times.

3. Add 50 μL diluted bacterial concentration in 4. Add in 100 µL of freshly prepared sterile TSB broth
respective wells using an appropriate multichannel (control well), ZnO-NPs suspended in TSB with test
pipette. Using a multichannel pipette is a faster concentrations and TSB containing the vancomycin in
and more efficient mean of adding the bacterial test concentration.
suspension into the wells.
5. Repeat the steps 5 to 10 from the above anti-adherence
4. Add sterile distilled water to the 4 corners of the assay protocol.
microplate to prevent evaporation of water from
6. Calculate the anti-biofilm activity of the test substance
the test wells. Evaporation of water in test wells
and vancomycin using the following formula:
can interfere with the results. Alternatively, the
microplate can be kept in a container placed with
moist filter paper during the incubation. Antibiofilm activity% =
Absorbance of control-Absorbance of test sample
× 100%
Absorbance of control
5. Cover the plate with the lid and place the plate in
an incubator at 37°C for 18 to 24 hours.
Method Validation
6. Take the 96-well plate out from incubator and
slowly remove the TSB either by decanting Determination of Biofilm Formation
or pipetting. Rinse the plate thrice with sterile
Based on the protocol, biofilm formation was indicated by
double distilled water and allow the plate to air
the violet stains. This show that TSB media was adequate
dry under the biosafety cabinet. Turn the plates
for biofilm formation whilst 0.1% (w/v) concentration of
upside down to hasten the process of drying.
crystal violet was sufficient for visible observation with the
Ensure it is dry before moving on to the next step.
naked eye and quantification by the spectrophotometer.
7. Dispense 100 µL of aqueous crystal violet (1%
w/v) into the test wells and let it stain the bacterial
cell walls for 10 to 15 minutes. Decant the crystal Assessment of Anti-adherence Assay
violet either into a sink or onto clean disposable
tissues. This protocol allows the determination of anti-adherence
8. Rinse the test wells three times with sterile double property of ZnO-NPs versus vancomycin using the 96-well
distilled water and allow the wells to dry under plate. The biomass of the bacterial cell was quantitatively
the biosafety cabinet. Alternatively, the plate can analyzed on the microplate reader at absorbance of 570 nm
be bathed subsequently with 3 dishes of water. showing a decreasing trend of biomass attachment with
increasing concentrations of ZnO-NPs tested. The result
9. Add 30% (v/v) glacial acid in water to solubilize show that ZnO-NPs at 65 μg/mL achieved a significant anti-
crystal violet and leave it standing for 15 minutes. adherence activity of 51.69 ± 2.55%. However, vancomycin
Ensure there is clear blue/violet solution with no at 0.5 μg/mL did not exhibit significant anti-adherence
visible residue in each of the test wells. activity when compared to negative control (TSB only)
(Figure 2).
10. Read the UV absorbance of all the wells at 570
nm (suggested range would be between 570 to

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 An Optimized Anti-adherence...

Figure 1. Schematic diagram shows the step-by-step protocol of the optimized ant-adherence and anti-biofilm assays. More detailed protocol should refer to the text.

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 Kemung HM et al.

Conflict of interest
The authors declare no conflict of interest.

Acknowledgement
This work was inspired by Monash PhD Research Training
Module which entitled “Bioprospective of microbes with
biopharmaceutical potential with bioinformatics and drug
discovery platforms” and financially by External Industry
Grants from Biotek Abadi Sdn Bhd (vote no. GBA-81811A)
and Monash Global Asia in the 21st Century (GA21)
research grant (GA-HW-19-L-01 and GA-HW-19-S02)
Figure 2. Anti-adherence assay using vancomycin as positive control and and Fundamental Research Grant Scheme (FRGS/1/2019/
MRSA ATCC 43300 as growth control. Experiment evaluated based on qua- WAB09/MUSM/02/1 and FRGS/1/2019/SKK08/
druplicate results with standard deviation. (n = 4, p < 0.05). *indicates signif- MUSM/02/7).
cant difference when compared to negative control (TSB only).

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