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In vitro susceptibilities of Candida species to Melaleuca alternafolia

In vitro susceptibilities of Candida and Aspergillus species to Melaleuca alternafolia (tea tree) oil

Jose A. Vazquez, Maria T. Arganoza, Dina Boikov, Julie K. Vaishampayan and Robert A. Akins

"Candida species are an important cause of opportunistic infection in the oral cavity of immunocompromised patients, especially HIV infected patients. Melaleuca oil obtained commercially was investigated since it is known to have broad antifungal properties. The in-vitro susceptibilities of Aspergillus and susceptible and resistant Candida species were performed utilizing serial dilutions in microtiter plates with Sabouraud dextrose agar and the commercial preparation of Melaleuca. As a comparator, in vitro susceptibilities to amphotericin B and fluconazole were also determined using the broth microdilution technique. The results demonstrate that Melaleuca inhibited the Candida species. However, the growth of Aspergillus was not inhibited at the concentrations tested. Thus, preparations containing Melaleuca alternafolia may be a useful alternative for superficial candidal infections. In fact, it may be a useful alternative regimen for advanced HIV-positive patients with oropharyngeal candidiasis refractory to fluconazole."

Summary

Candidaspecies are an important cause of opportunistic infection in the oral
cavity of immunocompromised patients, especially HIV infected patients.
Melaleuca oil obtained commercially was investigated since it is known to have
broad antifungal properties. The in-vitro susceptibilities of Aspergillus and sus-
ceptible and resistant Candidaspecies were performed utilizing serial dilutions in
microtiter plates with Sabouraud dextrose agar and the commercial preparation
of Melaleuca. As a comparator, in vitrosusceptibilities to amphotericin B and flu-
conazole were also determined using the broth microdilution technique. The
results demonstrate that Melaleucainhibited the Candida species. However, the
growth of Aspergilluswas not inhibited at the concentrations tested. Thus, prepa-
rations containing Melaleucaalternafolia may be a useful alternative for superfi-
cial candidal infections. In fact, it may be a useful alternative regimen for
advanced HIV-positive patients with oropharyngeal candidiasis refractory to flu-
conazole. However, controlled clinical studies to evaluate its efficacy are still nee-
ded.

Oropharyngeal candidiasis develops in 80-95% of
patients with AIDS [1,2]. The pathogenesis of this see-
mingly innocuous disease is very complex. Until now,
Candida albicanshas accounted for virtually all mucosal
candidiasis. Recently, however, other species such as
Candida glabrata, Candida parapsilosis, Candida
tropicalisand Candida krusei have caused serious symp-
tomatic oropharyngeal candidiasis (OPC), and on occa-
sion it may also be associated with esophageal
candidiasis. The oral azole antifungals clotrimazole, keto-
conazole, fluconazole and itraconazole are frequently used
in patients who are HIV-positive as initial or suppressive
therapy for oropharyngeal and esophageal candidiasis.
Unfortunately, the incidence of fluconazole-refractory
OPC is becoming increasingly more common and fre-
quently may emerge during therapy in advanced HIV-
positive patients [3,4]. Many of these patients they may
suffer from frequent clinical relapses despite high doses of
fluconazole and require parenteral amphotericin B. These
overwhelming infections frequently impair the quality of
life and may result in a reduction of fluid or food intake.
In searching for newer and less toxic compounds,
we have evaluated the oil of melaleuca. The oil was origi-
nally obtained from the leaves of a paperbark tea tree
grown in the central coastal region of eastern Australia.
Penford initially discovered the therapeutic value in 1922,
when he discovered antibacterial and antifungal properties
related to Melaleuca. Several of the active ingredients of
the tea tree oil include terpinen-4-ol and alpha-terpineol
[5-7]. Several investigators have recently evaluated its in-
vitro activity against Staphylococcus aureus, Escherichia
coli, Pseudomonas aeruginosa, Malassezia furfur,
Fusobacterium spp, Bacteroides spp, Prevotella spp, and
C.albicans[6-15].
In this study, we evaluate the in-vitro activity of
Melaleuca oil against Aspergillus species and known
resistant Candidaspeciesthathavebeenisolatedfromeither
advancedHIV-positivepatientssufferingfromfluconazole
andamphotericinBrefractoryOPCorfromimmunocompro-
misedpatientswithdisseminatedfungalinfections.
MATERIALS AND METHODS
Fungal strains. The organisms included clinical
specimens recovered from patients with candidemia,
OPC, esophageal candidiasis, or asymptomatic coloniza-
tion. The distribution of species included 50 C. albicans
isolates, 21 C. glabrataisolates, 10 C. tropicalisisolates,
seven C. parapsilosis isolates and five isolates each of
C. krusei, Candida lusitaniae, Candida kefyr, and
Candida guilliermondii. The quality control strains inclu-
ded C. albicans ATCC 90028, C. parapsilosis ATCC
90018, and C. glabrata ATCC 90030. In addition, five
isolates of Aspergillus fumigatus and five isolates of
Aspergillus nidulans were also evaluated.
In-vitro susceptibility analysis. Amphotericin B
and fluconazole were obtained from their respective
manufacturers. Oil of melaleuca T36-C7 (Tea-tree oil)
was obtained from Melaleuca Inc., Idaho Falls, Idaho,
U.S.A. This formulation contains 36% Terpinen 4-ol and
less than 10% 1,8 cineole as determined by gas liquid
chromatography. The MICs of all of the antifungal agents
for all of the isolates were determined in accordance with
the National Committee for Clinical Laboratory Standards
M27-A by a microdilution method [16].
A standard inoculum of Candida was diluted to a
final concentration of 1x102to 5x102 CFU/well in microti-
Aspergillus inoculum was prepared by suspending
Aspergillusconidia in buffered-saline. The conidia were
counted by a hemocytometer and then diluted to a concen-
tration of 106 conidia/ml [17]. Controls were grown on
drug-free and one drug containing media.
Candida and Aspergillus species were tested
against doubling dilutions of the oil of melaleuca [range,
2%-0.03 % (v/v)] as previously published by Hammer et
al. [11,12] prepared in RPMI in a 96-well microtiter plate.
Tween 80 (Sigma, St Louis, Mo.) was added at a final
concentration of 0.001% (v/v).
The MICs for amphotericin B and melaleuca were
defined as the lowest concentration that inhibited 100% of
the visible growth. The MICs of fluconazole was defined
as the lowest concentration that inhibited 80% of visible
growth when compared with the growth control. The data
are reported as the concentrations of each antifungal agent
necessary to inhibit 50% (MIC50) and 90% (MIC90) of the
isolates evaluated. All assays were done in duplicate to
verify the results. Since there are no definitive MIC break-
points that separate resistant from susceptible strains, we
used an MIC of >16 mg/ml to define fluconazole resis-
tance.
The MFC (mean fungicidal concentration) were
determined by subculturing 0.1 ml from the first microti-
ter well demonstrating complete growth inhibition and
from all wells with no visible growth onto Sabouraud dex-
trose agar plates that were incubated at 30C for 72 hours.
Afterwards, colonies were counted, and the MFC was
defined as the lowest concentration at which 99% of the
initial inoculum was killed.
RESULTS
Melaleuca oil had the lowest MICS50 and the
lowest ranges against C. albicans, C. parapsilosis and
C. kefyrwith a range of 0.06 - 0.25 % (Table 1). The most
susceptible of all of the Candida species is still
C. albicans, with an MIC range of 0.06 0.25%, an
MIC50 of 0.12%, an MIC90 of 0.25%, and an MIC50 of
0.50% (Table 1). The C. albicansstrains included 10 iso-
lates for which the MIC50 of fluconazole was 32μg/ml
and theMIC90 was 64 mg/ml. The MICS50 of melaleuca
for C. albicansisolates for which fluconazole MICs were
>16 μg/ml or <8 μg/ml were the same (0.12 %) (Table
2). The second most susceptible group of yeast isolates
includes the five C. lusitaniae and fiveC. guilliermondii
that have an MIC50 of 0.25% to melaleuca, with a similar
MIC range of 0.12 0.25%. C. krusei andC. tropicalis
Table 1. In-vitro susceptibilities of Candida albicans, non-albicans Candida spe-
cies and Aspergillusspecies to Melaleuca oil using broth microdilution assays.
______________________________________________________________
MIC (% vol/vol) MFC (%vol/vol)
_______________________________________
Organisms (No. tested) Range 50% 90% 50%
______________________________________________________________
C. albicans (50) 0.06 0.25 0.12 0.25 0.50
C.glabrata(21) 0.25 0.50 0.25 0.50 0.50
C. tropicalis (10) 0.12 0.50 0.25 0.50 0.50
C. parapsilosis(7) 0.06 0.25 0.25 - 0.50
C. kefyr (5) 0.06 0.25 0.25 - 0.50
C.krusei(5) 0.12 0.50 0.5 - 0.50
C. lusitaniae (5) 0.12 0.25 0.25 - 0.50
C. guilliermondii(5) 0.12 0.25 0.25 - 0.50
Aspergillus fumigatus(5) NI > 2.0 - NI
Aspergillus nidulans (5) NI > 2.0 - NI

have very similar MICS50 of 0.5 and 0.25%, respectively,
and an MIC range of 0.12 0.5% for both species. The
least susceptible of the Candida species to melaleuca were
the C. glabrata, with an MIC range of 0.25 0.50 %, an
MIC50 of 0.25%, and an MIC90 of 0.50% (Table 1). As
was the case with C. albicans, the MICS50of melaleuca for
the strains of C. glabratafor which fluconazole MICs
were >16 μg/ml or <8 μg/ml were similar at 0.25% and
0.12% for both groups, respectively (Table 2).
Unlike, the activity detected against the Candida
species; the melaleuca oil had essentially no effect against
any of the Aspergillus isolates we tested.
DISCUSSION
The results of this study confirm the excellent in
vitroefficacy of the tea tree oil Melaleuca, against the
more common Candida species. Melaleuca oil (tea tree
oil) is an old over the counter remedy with that possesses
potent in-vitro antifungal activity against a broad spec-
trum of Candidaspecies.
Melaleuca demonstrates the lowest MICs and is the
most active against C. albicans, C. kefyr, and
C. parapsilosis, with similar MICS50and narrow MIC ran-
ges. Melaleuca also has similar activity against C. lusita-
niae, C. guilliermondii and C. tropicalis. On the other
hand, melaleuca demonstrates less activity against
C. glabrata although still within the efficacy range, and
not much higher than the MICs for the very susceptible
strains of Candida. Moreover, the MIC and MFC results
indicate that melaleuca is fungicidal for all of the Candida
species evaluated, including those Candidaspecies that
were fluconazole resistant.
In addition to the broad anti-candidal activity of
melaleuca oil, the most exciting observation was the
remarkably good activity it demonstrated against the
strains of C. albicans and C. glabrata for which flucona-
zole MICs were high. These putatively resistant strains
were collected from patients with clinical failure to res-
pond to high dose fluconazole (1.2- 1.5 g/day). Essentially
the same melaleuca concentration was demonstrated for
both the putatively fluconazole-susceptible and fluconazo-
le-resistant strains of C. albicans and C. glabrata.
Moreover, melaleuca also demonstrated good activity
with low MICs against several Candidaspecies for which
the MICs of fluconazole were high.
Unfortunately, melaleuca oil demonstrated poor in
vitro activity against the two filamentous fungi,
A. fumigatusand A. nidulans.
In summary, the oil of melaleuca demonstrates
great potential as a novel antifungal compound with
potent in-vitro fungicidal activity against C. albicans,
C. glabrata, C. tropicalis and C. parapsilosis, the four
most commonly isolated species causing disseminated and
mucocutaneous candidiasis in the United States [1,3].
Melaleuca compounds may be a valuable addition to the
management of bacterial and fungal infections in the futu-
re [13,15,18,19]. In addition, because of its excellent in
vitroactivity against azole-resistant strains of C. albicans,
C. glabrata, and C. krusei including the multi-azole resis-
tant strains which have been recovered from AIDS
patients, melaleuca should be particularly useful for the
management of these clinically resistant candidal infec-
tions.
Recently, we published a small prospective study
evaluating a melaleuca based oral solution in patients with
AIDS and fluconazole-refractory oropharyngeal candidia-
sis [20]. At the 4-week evaluation, eight of the 13 patients
enrolled showed a significant response. Additionally,
seven out of 12 patients also demonstrated a significant
mycological response rate with a decrease in the colony
counts of Candidaspecies recovered during follow-up.
In addition, comparison of our data with previously
published data by Hammer et al. and Concha et al.
demonstrate very similar in vitrosusceptibility results
[10,11]. Unfortunately, as previously stated by Hammer et
al., it is difficult to compare data from different investiga-
tors since the chemical composition of the oils may be dif-
ferent, as well as the methodology of the studies [11].
We feel that the results of the in-vitro assays and
the small clinical study are extremely promising and that
further large, comparative prospective clinical studies are
warranted to determine the efficacy of the melaleuca com-
pounds for multi-drug resistant thrush. Especially in the
population of HIV-positive patients with advanced disease
who suffer from repeated episodes of mucosal candidiasis.
Table 2. Comparison of the in-vitro susceptibility of azole-susceptible and -resistant strains of Candidato melaleuca oil, amphotericin B and fluconazole.
______________________________________________________________________________________________________________________________
MIC MFC
______________________________________________________________________
Organisms (no. tested) Antifungal agent Range 50% 90% 50%
______________________________________________________________________________________________________________________________
C. albicans -S a(40)
Melaleuca oil (% vol/vol) 0.06 - 0.25 0.12 0.25 0.50
Amphotericin (μg/ml) 0.01 - 0.8 0.10 0.20
Fluconazole (μg/ml) 0.062 - 8 0.12 2
C. albicans-R a(10)
Melaleuca oil (% vol/vol) 0.06 - 0.25 0.12 0.25 0.50
Amphotericin (μg/ml) 0.02 - 0.40 0.05 0.20
Fluconazole (μg/ml) 16 - 64 32 64
C. glabrata-S b(10)
Melaleuca oil (% vol/vol) 0.12 - 0.50 0.12 0.25 0.50
Amphotericin (μg/ml) 0.05 - 0.80 0.20 0.80
Fluconazole (μg/ml) 0.50 - 4 1 8
C. glabrata-R b(7)
Melaleuca oil (% vol/vol) 0.25 - 0.50 0.25 - 0.50
Amphotericin (μg/ml) 0.20 - 0.40 0.40 -
Fluconazole (μg/ml) 16 - 64 32 -
______________________________________________________________________________________________________________________________
a
C. albicanssusceptible (S) and resistant (R) isolates, the MICS90of fluconazole were 2 and 64 μg/ml, respectively. bC. glabratasusceptible and resistant isolates, the MICS50of flucona-
zole were 1 and 32 μg/ml, respectively.

Full Article:
http://www.reviberoammicol.com/2000-17/060063.pdf





Keywords: Melaleuca alternafolia Candida In vitrosusceptibility

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