The Journal of the American Nutraceutical Association www.ana-jana.org
Vol. 6, No. 3, Summer 2003 Reprint
A Peer-Reviewed Journal on Nutraceuticals and Nutrition
Mark Houston, MD
Antimicrobial Effects of Plant-Derived
Essential Oil Formulations
on Pathogenic Bacteria
Robert A. Settineri, MS,1 Stuart M. Krassner, PhD2 Anne Vermilye, MNSh,3*
1 Sierra Productions, Research Division, Irvine, California
2University of California, Irvine, Irvine, California
3 Bio Excel, LLC®, Researcher, Mill Valley, California
775 E. Blithedale Ave., Suite 337
Mill Valley, California 94941
Antimicrobial disk susceptibility tests serve as standard
assays for measuring the activity of compounds against pathogenic
bacteria. In the current study, two plant-derived proprietary
essential oil blends were tested for their antibacterial
activity against five common strains of pathogenic bacteria
using disk susceptibility tests. A formulation intended for
topical use (EOF 1) inhibited the growth of Escherichia coli,
Klebsiella pneumoniae, andStaphylococcus aureus as evidenced
by zone inhibition diameter measurements when
compared to those reported for standard antibiotics. EOF 1
exhibited no activity against Proteus vulgaris and
Staphylococcus epidermidis. The second formulation (EOF
2), intended for inhalation use, inhibited the growth of all
five test bacteria strains with zone inhibition diameters two
to three times greater than those reported for standard antibiotics.
The growth of all five bacteria strains was inhibited
when a cotton swab impregnated with EOF 2 was suspended
above the bacterial lawn, indicating a true vapor or fume
effect by this formulation.
Key words: essential oils, antibacterials, disk susceptibility
testing, zones of inhibition, aromatherapy, botanical oil extracts, antimicrobials.
The antimicrobial properties of some plant-derived
essential oils have been recognized for hundreds of years1,2
and have been documented in scientific studies.3-7 It has
been demonstrated that the antimicrobial activities of one
natural oil, tea tree oil, obtained from Melaleuca alternifolia,
are attributable to its hydrocarbon and terpine constituents,
including terpinen-4ol, α-terpineol and linalool.8
The purpose of this study was to determine the
inhibitory effect of two botanical combinations of essential
oils against five common and clinically significant bacterial
pathogens. Antimicrobial activity was assayed by the
standard method adopted from the National Committee on
Clinical Laboratory Standards (NCCLS) for antibiotic susceptibility
One of the two proprietary formulations (EOF 1) Dental Delight was
developed for topical use, in the mouth and the other (EOF 2) Breathe Great for inhalation.
Besides the direct contact disk sensitivity method we devised a technique for the inhalation
formulation where a cotton swab was suspended above the agar-based bacterial lawn,
mimicking a true vapor or aroma effect.
MATERIALS AND METHODS
The following strains of gram negative and gram positive
bacteria were purchased from American Type Culture
Collection (ATCC) Manassas, VA: Escherichia coli
ATCC‚® 25922, Klebsiella pneumoniae ATCC‚® 27736,
Staphylococcus aureus ATCC‚® 25923, Proteous vulgaris
ATCC‚® 5380, and Staphylococcus epidermidis ATCC‚®
12228. Each strain was plated out on blood agar plates and
incubated for 18 hours at 35°C. Three to five identical 28
colonies from each agar plate were lifted with a sterile wire
loop and transferred into a tube containing 5 mL of tryptic
soy broth (TSB). Turbidity of each bacterial suspension was
adjusted with TSB media to reach an optical comparison to
that of a 0.5 McFarland standard, resulting in a suspension
containing approximately 1 to 2 x 108 CFU/mL.
A Wickerham Card (Hardy Diagnostics, Santa Maria, CA) was
used for the visual comparison.11
Within 15 minutes after adjusting the turbidity of the
inoculum suspension, Mueller-Hinton agar plates were
inoculated by streaking the swab over the entire sterile agar
surface. This procedure was repeated by streaking two
more times, rotating the plate approximately 60° each time
to ensure even distribution of the inoculum. As a final step,
the rim of the agar was also swabbed.
Susceptibility Disk Method
The essential oil formulations were acquired from Bio Excel, LLC, Mill Valley, CA., manufactured and formulated by, Bio Excel and Anne Vermilye, Mill Valley, CA. Both formulas contain a combination of
, essential oils that are plant-derived oil extracts (see Table 1). Fifteen microliters of
either tryptic soy broth (Control), or EOF 1, or EOF 2 was
placed on separate 0.25-inch blank filter paper disks (Hardy
Diagnostics, Santa Maria, CA.) The disks were dispensed onto
the surface of the inoculated agar plates and incubated at 35°C
for 18 hours. The diameters of the zones of complete inhibition
were recorded and each Petri dish were photographed.
Suspended Cotton Swab Method
Sterile cotton swabs were dipped in either sterile water
for control plates or in EOF 2 and pressed firmly against the
sides of the vessels to prevent dripping. They were then taped
to the lid of each Petri dish. The swabs were suspended above
the agar to avoid contact with its surface so that only the
volatile components of EOF 2 elicited inhibitory activity.
Figure 1. Susceptibility of Bacteria to Essential Oils
Each 8 mm sensitivity disk was impregnated with 15 μ l of either sterile TSB media
Figure 2. Bacteria Susceptibility to Essential Oils
to access new and complementary approaches to antibiotic
therapy. This screening study in the laboratory indicates
that essential oils may be considered to be used in combination
with standard topical and antibiotic therapies.
However, to verify clinical utility, it is necessary to extend
this research to human applications against similar strains
of pathogens and examine dose responses of both topical
and inhalation forms of the oils as well as testing various
modes of administration. Because of minimal, if any, toxicity12
and pleasant odor, these oils may have the advantage
of greater acceptance by patients and the community.
This initial antimicrobial screen also warrants further
studies with these formulations on antibiotic-resistant
strains and other pathogens such as viruses, fungi, mycoplasma, chlamydia, and yeasts.
The zones of bacterial inhibition were measured to the
nearest whole millimeter. The results of susceptibility disk
tests showed moderate zone inhibition with three of the five
bacterial strains in the EOF 1-treated cultures. Inhibition
diameters in this group ranged from 11 to 18 mm. All five
bacterial cultures treated with EOF 2 exhibited appreciable
increases of zones of inhibition ranging from 25–68 mm.
Both groups resulted in irregular zone patterns, unlike those
typically seen in standard antibiotic susceptibility tests.
Antibiotics, for the most part, present a clean circular edge
within the bacterial lawn, whereas the essential oil formulations
produced an asymmetric jagged or spiked edge (see Fig 1).
This effect may be a result of the pattern of radial
diffusion penetration through troughs of uneven growth distribution
within the bacterial lawn, giving rise to micro
channels of less dense or sparse bacterial accumulation.
The suspended swab method, incorporating EOF 2,
resulted in zones of inhibition ranging from 26–36 mm in
diameter in all five bacterial strains studied. An elliptical
pattern mimicking the shape of the cotton impregnated with
EOF 2 was observed (see Fig. 2). There appeared to be a
concentration gradient of clear pronounced inhibition in the
center, gradually feathering and fading to the periphery of
the zone in four of the five bacterial strains tested.
Presumably, the highest concentration of vapor molecules
was at the center of the swab. This feathered zone edge
effect was not prominent with Staphylococcus aureus.
The range of sensitivity to a wide variety of antibiotics by
strains of bacteria similar to those used in this study (13 – 29
mm with concentrations of antimicrobials ranging from 1 μg
to 350 μg per disk content) is documented in the NCCLS
“tables of zone diameter interpretive standards and equivalent
minimal inhibitory concentration (MIC) breakpoints.”9
In this screening study, the bacteriostatic and/or bactericidal
activities of two combination essential oil formulations
(EOF 1, EOF 2) were observed against five ubiquitous
strains of both gram negative and gram positive pathogenic
bacteria. EOF 1 is intended for topical use and showed minimal
to moderate antibacterial action comparable to the
lower zone inhibition ranges reported for most antibiotics,
11–18 mm versus 13–29 mm respectively. Escherichia
coli, Klebsiella pneumoniae and Staphylococcus aureus
were more sensitive to EOF 1 as evidenced by 15 mm, 16
mm, and 18 mm inhibited zone diameters respectively.
Insensitivity was observed with Proteous vulgaris and
Staphylococcus epidermidis with 11mm and 12mm zone
diameters respectively, when compared to NCCLS-reported
antibiotic minimal inhibitory range diameters.
EOF 2 is targeted for inhalation use. When the disk
method was utilized, the inhibition zone diameters for all
bacterial strains were approximately two to three times the
diameter of those of NCCLS-reported antibiotic sensitivity
tests, 25–68 mm compared to 13–29 mm respectively. The
suspended swab method more closely paralleled the intended
inhalation use of EOF 2 by permitting only the gaseous
phase of the formulation to make contact with the bacteria
hence giving meaning to so called, aromatherapy. The inhibition
seen by EOF 2 is greater than twice the reported
antibiotic inhibitory diameters (26–36 mm) and is noteworthy
since only a small concentration of the vapor molecules
would probably come in contact with the growing bacteria.
The inhalation application of EOF 2 may prove useful in the
prevention and/or treatment of upper respiratory infections
caused by some strains of bacteria, or in the treatment of
viral-induced secondary bacterial infections.
With the increase of worldwide bacterial resistance of
many strains of disease-producing bacteria, there is a need for further rsearch.
Figure 1. Susceptibility of Bacteria to Essential Oils
for controls or 15 μ l of each essential oil formula.
Table 1. Essential Oil Formulation Ingredients.
Thymus vulgaris linalool
Helichrysum itallicum serot
Rosmarinus officinalis camphora
Thymus vulgaris thymol
Table 2. Bacterial Zone of Inhibition Diameters with
Essential Oil Formulations
*nearest whole millimeter
Zone Diameter (mm)*
Bacteria EOF 1 EOF 2
Escherichia coli 15 45
Staphylococcus epidermidis 12 25
Staphylococcus aureus 18 47
Klebsiella pneumoniae 16 55
Proteous vulgaris 11 68
Table 3. Bacterial Zone of Inhibition Diameters with EOF 2
Suspended Swab Test, Breathe Great
Bacteria Zone Diameter (mm)*
Proteous vulgaris 26
Klebsiella pneumoniae 25
Escherichia coli 32
Staphylococcus epidermidis 25
Staphylococcus aureus (MRSA) 36
1. Potterton D, Shellard EJ, Press JR. Culpeper’s Colour Herbal,
2d ed. . London, England: Foulsham 1996.
2. Lee K. Herbal success in MSRA treatment. Hospital Doctor.
3. Celso VN, et al. Antibacterial activity of Occimum gratissimum
L. essential oil. Mem Inst Oswaldo Cruz, Rio de Janiero.
4. Jedlickova Z. Antibacterial properties of Vietnamese cajeput oil
and occimum oil in combination with antibacaterial agents. J
Hyg Epidemiol Immunol. 1992;36:303-309.
5. Lima EO, et al. In vitro antifungal activity of essential oils
obtained from officinal plant against dermatophytes. Mycoses.
6. Nwosu JO, et al. Preliminary studies of the antifungal activities
of some medical plants against Basidiobolus and some other
pathogenic fungi. Mycoses. 1995;38:191-195.
7. Ramanoelina AR, et. al Antibacterial action of essential oils
extracted from Madagascar plants. Arch Inst Pasteur
8. Carson CF, et al. Susceptibility of methicillin-resistant
Staphylococcus aureus to the essential oil of Melaleuca alternifolia.
Antimicrobial Chemother. 1995;35:421-424.
9. Performance Standards for Antimicrobial Disk Susceptibility
Tests, 5th ed. M2-M5. Villanova, Pa: National Committee for
Clinical Laboratory Standards (NCLS). 1930.
10. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility
testing by a standardized single disk method. Am J
Clin Pathol. 1966;45:493-496.
11. Carlberg D. Determining the effect of antibiotics on bacterial
growth by optical and electrical methods. Antibiotics in
Laboratory Medicine, 2d ed. Philadelphia, Pa: Williams and
12. Tisserand R, Balacs T. Essential Oil Safety. Churchill
Livingstone, Medical Division of Pearson Professional Limited.
New York, NY. 1996:228-229
JOURNAL OF THE AMERICAN NUTRACEUTICAL ASSOCIATION
A Peer-Reviewed Journal on Nutraceuticals and Nutrition
Mark Houston, MD – Associate Clinical Professor of
Medicine, Vanderbilt University School of Medicine;
Director, Hypertension Institute and Vascular Biology,
Saint Thomas Medical Group, Saint Thomas Hospital,
Jordan Asher, MD – Co-Founder, Hypertension Institute,
Saint Thomas Medical Group - Saint Thomas Hospital,
Assistant Clinical Professor of Medicine, Vanderbilt
University School of Medicine, Nashville, Tennessee.
Ethan Basch, MD, MPhil – Fellow in Hematology/Oncology,
Memorial Sloan-Kettering Cancer Center, New York, Chief
Editor, Massachusetts General Hospital Primer of Outpatient
Jan Basile, MD – Associate Professor of Medicine, Ralph
H. Johnson VA Medical Center, Medical University of
South Carolina, Charleston, South Carolina.
Russell Blaylock, MD – Clinical Assistant Professor,
University of Mississippi Medical Center, Jackson,
Hyla Cass, MD – Assistant Professor of Psychiatry, UCLA
School of Medicine, President, The Healthy Foundation -
Vitamin Relief - USA/Children First. Los Angeles, California.
Lisa R. Colodny, PharmD, BCNSP – Clinical
Coordinator, Broward General Medical Center, Ft.
Loren Cordain, PhD – Professor, Department of Health
and Exercise Science, Colorado State University, Ft.
Jeanette Dunn, EdD, RN, CNS – Former Associate Dean
of Nursing, University of Tennessee. Co-director,
Foundation for Care Management, Vashoon, Washington.
Brent Eagan, MD – Professor, Pharmacology and
Medicine, Medical University of South Carolina,
Charleston, South Carolina.
Christopher M. Foley, MD – Medical Director,
Integrative Care, St. Paul, Minnesota. Serves on the teaching
faculty, University of Minnesota, School of Pharmacy.
Clare M. Hasler, PhD – Assistant Professor of Nutrition,
Department of Food Science and Human Nutrition,
University of Illinois at Urbana-Champaign.
Ralph G. Hawkins, MD, FRCPC – Associate Professor of
Medicine, Division of Nephrology, University of
Robert Krueger, PhD – Professor of Pharmacognosy,
School of Pharmacy, Ferris State University, Big Rapids,
Alexander Mauskop, MD, FAAN – Director, New York
Headache Center, and Associate Professor of Clinical
Neurology, State University of New York (SUNY),
Downstate Medical Center, Brooklyn, New York.
Mark J.S. Miller, PhD – Research Professor and Director
of Pediatric Laboratories, Department of Pediatrics, Albany
Medical College, Albany, New York.
Garth L. Nicolson, PhD – President, Chief Scientific
Officer and Research Professor, The Institute for Molecular
Medicine, Huntington Beach, California.
Anthony J. Silvagni, DO, PharmD, MSc, FACOFP –
Dean, College of Osteopathic Medicine, Nova
Southeastern University, Ft. Lauderdale, Florida.
Catherine Ulbricht, PharmD – Senior Attending Pharmacist,
Massachusetts General Hospital. Adjunct Clinical Professor,
Massachusetts College of Pharmacy, Boston, Massachusetts.
Editor-in-Chief, Journal of Herbal Pharmacotherapy.
C. Wayne Weart, PharmD, BCPS, FASHP – Professor
and Chair, Department of Pharmacy Practice, and Associate
Professor, Department of Family Medicine, Medical
University of South Carolina, Charleston, South Carolina.
Bernd Wollschlaeger, MD – Board-certified family practice;
Assistant Clinical Professor of Medicine and Family
Medicine, University of Miami, School of Medicine.