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#1 2021-04-12 18:43:30

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From: Bellingham
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Essential oils Eradicate Pseudomonas and Staphylococcus a.

Selected Antimicrobial Essential Oils Eradicate Pseudomonas spp. and
Staphylococcus aureus Biofilms
Nicole L. Kavanaugh and Katharina Ribbeck
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
Biofilms are difficult to eliminate with standard antimicrobial treatments due to their high antibiotic resistance relative to freeliving cells. Here, we show that selected antimicrobial essential oils can eradicate bacteria within biofilms with higher efficiency
than certain important antibiotics, making them interesting candidates for the treatment of biofilms.
Microbial biofilms pose a challenge in clinical and industrial
settings where the need for sterility is paramount. Bacteria
within biofilms are more resistant to antibiotics and disinfectants
than individual cells in suspension (6, 25). Several mechanisms
can account for the increased antibiotic resistance in biofilms,
including the physical barrier formed by exopolymeric substances
(14), a proportion of dormant bacteria that are inert toward antibiotics (15), and resistance genes that are uniquely expressed in
biofilms (17, 19, 16, 27). Together, these bacterial features that
create resistance to antibiotics drive the need for novel strategies
that will effectively kill bacterial biofilms.
Plant essential oils have been used for hundreds of years as
natural medicines to combat a multitude of pathogens, including
bacteria, fungi, and viruses (10). Several essential oils confer antimicrobial activity by damaging the cell wall and membrane, leading to cell lysis, leakage of cell contents, and inhibition of proton
motive force (4). In addition, there is evidence that they effectively
kill bacteria without promoting the acquisition of resistance (1,
22). Finally, many essential oils are relatively easy to obtain, have
low mammalian toxicity, and degrade quickly in water and soil,
making them relatively environmentally friendly (11).
Here, we probed the ability of selected essential oils to kill biofilms formed by Pseudomonas aeruginosa (PAO1), Pseudomonas
putida (KT2440), and Staphylococcus aureus SC-01. P. aeruginosa
is a Gram-negative bacterium found in soil, water, and animals,
but it is also an opportunistic pathogen in humans. It can infect
the pulmonary and urinary tracts, wounds, and burns and cause
devastating medical complications by forming biofilms on medical devices, such as catheters. The biofilms formed by P. aeruginosa
allow this pathogen to evade treatment with antibiotics and cause
persistent, sometimes deadly, infections. The closely related species Pseudomonas putida can also form biofilms, but it is not a
pathogen. Usually, P. putida is found in the environment, especially in soil, in freshwater, and on the roots of plants. The Grampositive species S. aureus can exist both as a commensal and as a
pathogen. As a pathogen, this bacterium is responsible for a broad
range of maladies, from superficial skin infections to serious systemic infections. Treatment of S. aureus is complicated by antibiotic resistance, which is especially problematic in multidrug-resistant strains such as methicillin-resistant S. aureus (MRSA).
Essential extracts from the bark of plants in the genus Cinnamomum have antibacterial activity toward a range of microbes,
including P. aeruginosa (2, 21, 24). In previous studies, the effect of
Cinnamomum extract on P. aeruginosa was tested against individual bacteria in solution. Here, we asked if this potent antimicrobial
would also be effective against this bacterium within a biofilm.
To address this question, P. aeruginosa biofilms were grown on
the air-liquid interface of a microscope slide, which was halfway
submerged in Mueller-Hinton broth (MHB) containing PAO1 at
an optical density at 600 nm (OD600) of 0.0025. After 24 h of
growth at room temperature, biofilms were washed with H2O and
then challenged with cation-adjusted MHB containing 0.2% or
0.1% (vol/vol) cassia oil (Cinnamomum aromaticum, 100% pure;
Aura Cacia) or 3 g ml1 colistin. In a separate assay, using the
CLSI broth microdilution method modified with a 2-hour challenge period (7), 0.2% (vol/vol) cassia oil and 3 g/ml colistin
were determined to be the lowest concentrations of these chemicals required to eradicate P. aeruginosa in solution (Table 1). In the
case of cassia oil, 0.1% (vol/vol) Tween 80 was added to mix the oil
with the medium (5). At this concentration, Tween 80 did not
affect the growth or viability of planktonic cells or cells in a biofilm
(data not shown). After 2 h, the treated biofilms were rinsed with
H2O, stained with LIVE/DEAD BacLight (Invitrogen), and imaged by wide-field fluorescence microscopy. BacLight uses a combination of two nucleic acid dyes: SYTO9, a membrane-permeable
green dye that labels both viable and dead cells, and propidium
iodide, a membrane-impermeative red dye that labels only membrane-compromised cells and eliminates the green SYTO9 signal.
Planktonic cells (final OD600  0.25) were challenged with the
same concentration of cassia oil or colistin used against the biofilms for 2 h and then placed into a glass-bottom 96-well plate for
Our results show that the MIC of colistin (3 g ml1
) needed
to eradicate planktonic cells was not effective against cells within a
biofilm, since a large fraction of the cells remained stained in green
(Fig. 1, top right). In contrast, the MIC of cassia oil against planktonic cells (0.2%) (Table 1) was also sufficient to kill the vast
majority of P. aeruginosa cells within a biofilm (Fig. 1, middle),
suggesting that these cells were not protected from cassia oil. A
slightly lower concentration of the essential oil (0.1%) did not kill
bacteria in solution or in biofilms (Fig. 1, bottom).
Received 8 November 2011 Accepted 20 March 2012
Published ahead of print 30 March 2012
Address correspondence to Katharina Ribbeck, ribbeck@mit.edu.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
June 2012 Volume 78 Number 11 Applied and Environmental Microbiology p. 4057– 4061 aem.asm.or


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