Candida Azole Antifungal Drugs

Journal of A pplied Sciences Res earch, 5(10): 1325-1334, 2009
© 2009, INSInet Publication
Increasing Susceptibility of Clinical Isolates of Candida Species to
Azole Antifungal Drugs
M.E. Zain, H.M. Mahdy and M.A. Hemdan
Botany & Microbiology Dept., Faculty of Science, Al-Azhar University, Cairo, Egypt.
Abs tract: Eleven Candida s trains is olated from clinical s pecimens were found to be re s is t ant to ketozole
a n d la mis il d rugs at concentration of 2.5 mg/ml. The effect of dis infectants , vitamins and nitrogenous
bas es on s u s ceptibility of Candida s trains to ketozole and lamis il were inves tigated. The s ens itivity of
Candida s trains to lamis il was s ignificantly inc re a s ed by s odium hypochlorite (5 ml/ml), potas s ium
hydroxide (5 mg/ml), potas s ium iodide (5 mg/ml), iodine (5 mg /ml), vitamin H (50 mg/ml), vitamin B1 2
(50 mg/ml), riboflavin (250 mg/ml) and folic acid (50 mg/ml). O n t he other hand, the s us ceptibility of
Candida s trains was s lightly affected by guanine, adenine and uracil. However, the re was no effect on
the s us ceptibility of Candida s trains to ketozole.
Key words : Candida, Res is tance to antifungal, A zole drugs .
INTRODUCTION
The e mergence of Candida s pecies as s ignificant
human pathogens has bee n part of a general increas e
in the number of infections caus ed by fungal pathogens
during the las t 20 years . This s hift in the epidemiolo gy
of fungal infections has been correlated with the
increas e in the number of immuno c o mpromis ed and
immunos uppres s ed patients , as well as with an increas e
in the us e of invas ive me dical procedures , in-dwelling
central
veno u s
catheters and broad-s pectrum
antibiotics [1].
W hile the introduction of azole a n tifungal agents
has improved the ou t c ome of many fungal infections ,
treatment failure due to res is tan c e t o thes e agents has
become a clinical problem. In particular, res is t a n c e to
azole drugs has been as s ociated with oral candidias is
relaps es in human immunodeficiency virus (H IV)infected and A IDS patients fo llowing extens ive
therapeutic and prophylactic us e of fluconazole [2-4].
A variety of molecular mechanis ms by whic h
Candida cells can develop res is tance to azole drugs
have been des cribed previous ly. A zole res is tance can
be caus ed by increas ed expres s ion of the target enzyme
(cytochrome P450 lanos terol 14 -demethylas e), res ulting
in increas ed levels of the enzyme in cells [5 ,6 ], or by
point muta t ions which reduce its affinity for azole
drugs [7 - 1 2]. Reduced ac c umulation of the drug due to
increas ed efflux is another mechanis m commonly
involved in azole res is tance in clinical Candida is olates
[1 3 -1 8 ]
. In addition, res is tance to a zo le drugs has als o
been as s ociate d w ith modifications of the ergos terol
bios ynthetic pathway, s uch a s defects in the s terol
C5,6-des aturation s tep [1 9 -2 2 ].
A decreas e in In Vitro s us ceptibility of C. albicans
to azoles was initially report e d in the late 1970s in
p a t ie n t s w it h c h ro n i c ( n o n - A u t o immu n e
polyendocrinopathy-candidias is -ectodermal dys trophy,
non- A PECED) mucocutan e o u s c andidias is with
repeated and prolonged cours es of thera p y [ 2 3 ,2 4 ]. C.
albicans with decreas ed in vitro s us ceptibility to azoles
ha v e been is olated from HIV patients [2 5 ]. However,
thes e interpretations are mainly bas ed on M IC data
from heterogeneous patient groups lacking clinical data.
Long-term data of homogeneous patient groups with
recorded antifungal us age d o e s not exis t. Rautemaa et
al. [2 6 ] reported the us age of antifungals in 23 Finnis h
A PECED p atients over the pas t 30 years and the
c oncomitant acquis ition of decreas ed s us ceptibility to
fluconazole in C. albicans.
W hile mos t is olates o f C. dubliniensis are
s us ceptible to fluconazole, a number of is olates
exhibiting either decre as ed s us ceptibility or res is tance
to fluconazole have been des cribed [2 7 -3 1 ]. Thes e is olates
were mos tly recovere d from HIV-infected patients
receiving fluconazole therapy. Furthermore, M oran et
al. [3 2 ] s howed t hat fluconazole res is tant derivatives
could be generated from s us cep t ib le is olates following
s equential expos ure to fluconazole in vitro, thus
indicating that C. dubliniensis has the ability to rapidly
develop res is tance to this drug. In vitro expos ure to
fluconazole has als o been s hown to re s ult in increas ed
a d h erence of C. dubliniensis to epithelial cells whic h
correlated wit h increas ed proteinas e s ecretion levels ,
whereas the adherence of C . albicans was decreas ed
under the s ame conditions [3 3 ]. It is thus pos s ible that
treatment with fluconazole could provide a s elective
Corresponding Author: M .E. Zain, Botany & M icrobiology Dept., Faculty of Science, Al-Azhar University, Cairo,
Egypt.
1325
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
advantage favouring the growth of C. dubliniensis over
C . albicans is olates in the oral cavity. This c o u ld
explain the high rec o very rate of C. dubliniensis
is olates from HIVinfected patients receiving fluconazole
treatment. In ad d it ion, Odds et al. [2 8 ] have s hown that
the geometric mean M ICs of C. dubliniensis for azole
drugs are s ignificantly higher than thos e of C. albicans.
Failure to accumulate azole antifungals has been
s hown to be a major factor involved in azole res is tance
in clinical C. albicans is olates , and numerous s t u dies
have repo rt e d the as s ociation of azole drug res is tance
wit h the up-regulation of genes encoding multidrug
efflux trans porters [1 7 ,3 4 ].
In this s tudy, the effect of dis infectants and
v itamins on s us ceptibility of clinical is olates o f
Candida to azole antifungal agents was determined.
MATERIAL AND METHODS
Is olation Media: The Cand i da s trains were is olated
and s ubcultured on Sabouraud’s agar medium (gradients
(g/L): glucos e, 4.0; yeas t extract, 4.0; malt extract, 10.0
and agar, 20.0. The pH was adjus ted at 7.1) a n d Yeas t
M alt extract agar medium (g ra dient (g/L): yeas t extract,
3.0; malt extract, 3.0; glucos e, 10.0 and agar, 20.0).
Identification of Candida S trains : For id entification,
the Candida s trains were cultured on Sabouraud’s agar
medium s upplemented with chloramphenico l, 0.5 g;
penicillin, 20 I. U./ml; s trep t o my c in, 4.0 g. The pure
culture was then trans ferred to minimal medium (25 ml
of s olution A [ammonium chloride, 20 g ; a mmonium
nitrate, 4 g; s odium s ulphate; potas s ium mono-hydrogen
phos phate; 12 g; potas s iu m d i-hydrogen phos phate, 4
g; magnes ium s ulphate, 0.4 g; dis tilled water, 1000 ml]
a n d 1 ml of s olution B [glucos e, 20 g; dis tilled water,
100]; A gar, 20 g).
S us ceptibility of Ca n d i d a s trains to azole drugs : The
purified is olates of Candida were grown on s abourau d
agar medium amended with Ketozole (ketokonazole)
200 mg/tablet , la mis il (terbinafine) 250 mg/tablet at
concentratio n of 2.5 mg/ml and a mixture of ketazole lamis il at concentration of 5 mg/ml (2.5 mg/ml, each).
Effe c t of D i s i n fe c tan ts a n d V i tami n s on
S u s ceptibility of Candida to Azole Drugs : Different,
commonly us ed, dis infectants and v itamins were
inves tigated for their effect on s us ceptibility of Candida
to ketozole and lamis il drugs . The res is tant Ca ndida
s trains were s ubcultured on minimal medium s e parately
s upplemented with the tes ted compound. The Candida
s trains were then tran s fe rred into a s abouraud medium
s upplemented with ketozole (2.5 mg/ml) lamis il (2.5
mg/ml) and mixture of ketozole and lamis il (5 mg/ml).
1326
RES ULTS AND DIS CUS S ION
Is olation of Yeas t S trains : Pathogenic yeas t s trains
were clinically collected from different infe c t ion s ites
and lab s amples o f patients with different s ex and age.
The diagnos t ic clinical s ymptoms of yeas t infections
were examined by phys icians . The obtained yeas t
s trains were s ubcultured and purified on Sabouraud and
Yeas t-M alt extract media.
The obtained yeas t is olates were s ubcu lt u red on
Saboura ud medium s upplemented, s eparately, with
ketozole, lamis il (at concentration of 2.5 mg/ml) and
ke t o zole – lamis il mixture (5 mg/ml; 2.5 mg/ml each).
Eleven yeas t s trains , res is ta n t to ketozole and lamis il,
were obtained and identified (Table 1).
Identification of the Yeas t S trains : The A PI 20 C
A UX yeas t identification s ys tem was us ed for
identification of the yeas t s trains . The is olated and pure
cultures of the y e a s t s trains were s ubcultured on
Sab o u ra u d 's a g a r me d iu m s u pplemented with
chloramphenicol, penicillin and s treptomycin prior to
identification proces s .
The yeas t identification s ys tem (A PI 20 C A UX)
confirmed that the eleven s trains are belongin g to four
s pecie s of the genus Candida, namely; C. glabrata (5
is o la t es ; 1, 2, 4, 5, 6), C. parapsilosis (3 is olates ; 7, 9,
10), C. albicans (2 is olates ; 3, 11) a n d C . famata (one
is olate; 8).
Eff e c t o f D i s i n fe c tan ts an d V i tami n s on
S us c e ptibility of Candida S trains to Azole Drugs :
The effect of dis in fe c t a n t s a n d v itamins on
s us ceptibility of Candida s trains to ketozole and lamis il
drugs were inves tigated in this s tudy.
The inves tigated res is tant s trains of Candida were
grown on minimal medium s upple mented with the
tes ted compound. Each s train was then trans ferred into
Sabouraud medium s upplemented with t he drug(s )
(ketozole, 2.5 mg/ml; lamis il, 2.5 mg/ml; and ketozolelamis il mixture, 5 mg / ml), s e p a ra t e ly , and its
s us ceptibility was determined.
The o b tained res ults revealed that there was a
remarkable effect for all the dis infectants and vitamins
us ed in this s tudy on s us ceptibility of Candida s trains
t o lamis il. However, there was almos t no effec t o n
s us ceptibility of Candida s trains to ketozole.
Dis infectants :
a. S odium hypochlorite: The effe c t of s odium
hypochlorite (5 ml/ml) on s us ceptibility of the Candida
s trains was determined (Table 2). T here was a
tremendous effect of s o d iu m h y p o c h lo rite on
s us ceptibility of the inves tigated Candida s t rains .
Sus ceptibility o f eight s trains were changed from
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
res is tant to s ens itive s trains to both lamis il and
ketozole-la mis il mixture. Only C. glabrata 1, C.
parapsilosis 7 and C. famata 8 were s till res is tant.
b. Potas s ium hydr oxide: Of all the inves tigated
s trains , only Candida parapsilosis 9 remained res is tant
to lamis il and ketozole-la mis il mixture after being
treated with potas s ium hydro xid e (5 mg/ml) (Table 3).
In addition, C. glabrata 2, 5 were remained res is tant to
lamis il.
However, all the s trains remained res is tant to
ketozole after being treated with potas s ium hydroxide.
c. Potas s ium Iodide : Candida albicans 3, 11; C.
g labrata 4, 5; C. parapsilosis 10 have been chan g e d
from res is tant to s ens itive s tra in s t o both lamis il and
ketozole-lamis il mixture (Table 4). M oreover, Candida
glabrata 2, 6; C. paraps i losis 9 were changed to
s ens itive s trains to ketozole-lamis il mixture.
d. Ethylene di ami ne tetra acetic acid (EDTA): The
res ults s howed that the EDTA has a great effect on the
s us ceptibility of the s trains to the antifungal drugs us ed
in this s tudy (Table 5).
The s us ceptibility of C. glabrata 1, 4, 5; C.
albicans 3, 11; C. parapsilosis 9, 10 were comple t e ly
changed from res is tant to s ens itive s trains to all th e
three antifungals us ed (ketozole, lamis il, ketozolelamis il mixture).
A lthough, C. glabra ta 6 and C. parapsilosis 7
have los t their res is tancy to lamis il and ketozole-lamis il
mixture.
e. Iodine: Des pite that there was 3 s trains ; namely, C.
glabrata 4, 6 and C. albicans 11 were not able to
grow on minimal medium a mended with iodine (5
mg/ml), all t h e other eight s trains were changed from
res is tant to s ens itive s trains to all the us ed antifungals
(ketozole, lamis il, ketozole-lamis il mixture) (Table 6).
II. Vitamins :
a. vitamin H (Biotin): The obtaine d res ults s howed
that the s us ceptibility of Candida s trains was affected
by vitamin H (Table 7). Candida glabrata 1 w a s
changed from re s is tant to s ens itive s train to ketozole
and lamis il drugs . On the other hand, Candida albicans
3; C. glabrata 4, 5 and C. p a r a p s i losis 10 s howed no
growth, became s ens itive, a t 2.5 mg/ml of lamis il and
5 mg/ml of ketozole-lamis il mixture.
b. Vitamin B 1 2 : The re w a s a remarkable effect of
v itamin B1 2 on s us ceptibility of the Candida s trains t o
ketozole and lamis il drugs (Table 8).
1327
The res ults revealed that the C a n d i d a glabrata 1
an d C . famata 8 were greatly affected by vitamin B1 2
s ince their s us ceptibility to ke tozole, lamis il, and the
mixture of ketozo le-lamis il has been changed from
res is tant to s ens itive. A lt h o u g h , C. glabrata 6 and C.
parapsilosis 9 were changed to s ens itive to ketozole.
M oreover, only 4 s trains , C. glabrata 6; C. parapsilosis
7, 9; and C. albicans 11 were remained res is tant to
lamis il a n d o n ly 3 s trains , C. glabrata 2, C.
parapsilosis 7 and C. albicans 11 were remained
res is tant to ketozole-lamis il mixture.
c. R i boflavin: The s us ceptibility of the inves tigated
Candida s trains to ketozole–lamis il mixture w as greatly
affected by riboflavin (Table 9). Only C a n d i da
glabrata 1 and 6 t h a t re ma ined res is tant to
ketozole–lamis il mixture, while all t h e o t her 9 s trains
were changed to s ens itive s trains . However, the
s us ceptibilit y of C. albicans 3, C. parapsilosis 7, and
Candida famata 8 t o lamis il was changed from
res is tant to s ens itive after treatment with riboflavin.
d. Folic acid: A fter t re atment the 11 res is tant Candida
s trains with folic acid, Candida glabrata 5 and C.
parapsilosis 9 s howed no growth, became s ens itive, on
medium containing ketozole (Table 10). W hile C.
glabrata 6 and C. famata 8 remained res is tan t to of
ketozole – lamis il mixture. Out of all s trains , C.
glabrata 6 was the on ly s t rain that remained res is tant
to all the inves tigated antifungals .
Trans formation of S ens itivity to Res is tant Candida
S trains : The res is tant C a n d i d a s t ra in s w e re
s u c c e s s fu lly grown u p o n min ima l me d iu m
s upplemented with s mall DNA fragment s o f s ens itive
Candida s trains as ca rb o n and nitrogen s ources
(table 11).
The res ults revealed that C. glabrata 1 and 5 were
changed to s ens itive to all the inves tigated azole drugs .
A dditionally, C. gla brata 2, 4 and C. albicans 3, 11
were changed to s ens itive s trains t o la mis il. On the
other h a n d , o n ly 3 s trains , C. glabrata 6, C. famata 8
and C. parapsilosis 10 remained res is tant to ketozolelamis il mixture.
Dis cus s ion: Candida albicans is t h e mos t pathogenic
Cand i d a s pecies and is the mos t common caus e of
Candida infectio n s . However, over the las t 2 decades ,
s ince th e introduction and wides pread us e of the azole
drugs fluconazole and itraconazole, other Candida
s pecies have emerged as s ignificant pat h o g e ns of
clinical importa nce [1]. In this s tudy, eleven is olates of
Candida were is olated from Candida infec t ions , only
two s trains were identified as Candida albicans.
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
Table 1: T he growth of Candida strains Sabouraud medium supplemented ketozole and lamisil drugs.
Strain No.
Yeast strain
Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml)
Ketozole– Lamisil (5 mg/ml)
1
C. glabrata
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2
C. glabrata
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3
C. albicans
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4
C. glabrata
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5
C. glabrata
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6
C. glabrata
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7
C. parapsilosis
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------8
C. famata
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------9
C. parapsilosis
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------10
C. parapsilosis
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------11
C. albicans
+ve
+ve
+ve
+ve, growth observed (Resistant)
Table 2: Effect of sodium hypochlorite on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with sodium
Growth in presence of
hypochlorite (5 ml/ml)
--------------------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml)
Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
+ve
-ve
-ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
Table 3: Effect of potassium hydroxide on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with potassium
Growth in presence of
hydroxide (5 ml/ml)
--------------------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml)
Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
+ve
+ve
-ve
-ve
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
1328
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
Table 3: Continue
C. parapsilosis 7
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
+ve
-ve
-ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
Table 4: Effect of potassium iodide on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with potassium
Growth in presence of
iodide (5 mg/ml)
--------------------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml)
Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
+ve
-ve
-ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
Table 5: Effect of EDT A on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with EDT A (5 mg/ml)
Growth in presence of
----------------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml)
Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
-ve
-ve
-ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
1329
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
Table 6: Effect of iodine on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with iodine (5 mg/ml)
Growth in presence of
----------------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml)
Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
-ve
ND
ND
ND
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
-ve
ND
ND
ND
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
-ve
ND
ND
ND
+ve = growth (Resistant), -ve = no growth (Sensitive), ND = Not determined.
Table 7: Effect of vitamin H on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with (50 mg/ml)
Growth in presence of
----------------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml)
Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
+ve
-ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
+ve
+ve
+ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
Table 8: Effect of vitamin B 12 on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with vitamin B 12 (50 mg/ml)
Growth in presence of
-----------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml) Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
-ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
+ve
-ve
-ve
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
1330
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
Table 8: Continue
C. glabrata 6
+ve
-ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
-ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
+ve
+ve
+ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
Table 9: Effect of riboflavin on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with riboflavin (250 mg/ml)
Growth in presence of:
-----------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml) Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
+ve
+ve
-ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
Table 10: Effect of folic acid on susceptibility of Candida strains to azole drugs.
Yeast strain
T reatment with folic acid (50 mg/ml)
Growth in presence of:
-----------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml) Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
+ve
-ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
+ve
+ve
-ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
1331
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
Table 11: Growth of the resistant Candida strains after transformation of sensitive DNA fragments.
Yeast strain
T ransformant Candida strain
Growth in presence of:
-----------------------------------------------------------------------------------------------------Ketozole (2.5 mg/ml)
Lamisil (2.5 mg/ml) Ketozole – Lamisil (5 mg/ml)
C. glabrata 1
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 2
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 3
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 4
+ve
+ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 5
+ve
-ve
-ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. glabrata 6
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 7
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. famata 8
+ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 9
+ve
+ve
+ve
-ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. parapsilosis 10 +ve
+ve
+ve
+ve
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. albicans 11
+ve
+ve
-ve
-ve
+ve = Positive growth (Resistant), -ve = Negative growth (Sensitive).
It is now w e ll es tablis hed that long-term treatment
of Candida infections with azole drugs can res ult in
the development of antifungal res is tance. In particular,
fluconazole treatment of oral c andidias is in HIVinfected and A IDS pat ie n ts has been as s ociated with
treatment failures [2-4]. The res ults of the current s tudy
revealed that the s us ceptibility of fours s p e cies of
Candida to ketozole and lamis il drugs has been greatly
decreas ed.
The tre atment of Candida-related infections was
cons iderably enhanced b y t h e in t roduction of
fluconazole in 1990 [3 5 ], however, s ince the introduction
of azoles , an increas e in the prevalence of C . glabrata
and C. k rusei bloods tream is olates with reduced
s us ceptibilities to triazole antifungals has been reported,
and acquired azole res is tance of C. albicans is olates
( e . g . , in mu c o s a l d is e a s e s i n a c q u i r e d
immunodeficiency s yndrome) has become a major
concern [3 6 , 3 7 ] . The is olated s trains of Candida in this
s tudy were found to be belonging to four s pecies ; C.
glabrata, 5 is olates ; C. parapsilos i s , 3 is olates ; C.
albicans, 2 is olates and C. famata, one is olate.
In recent years , in t eres t has grown in dis covering
new antimicrobial drugs in res pons e to t h e ever
increas ing incidence of advers e s ide effects as s ociated
with conventional drugs and the emerge n c e of
res is t a nce to antibiotics , s ynthetic dis infectants and
germicides [3 8 ]. The res ults of this s tud y revealed that
the s us ceptibility of Candida s pecies to azole antifungal
agents can be greatly increased by certain dis infectants
and vitamin s . It s eems to be that thos e compounds
affect the metabolis m of the res is tant s trains .
1332
REFERENCES
1.
2.
3.
4.
5.
6.
M o ran, G.P., D.J. Sullivan and D.C. Coleman,
2002. In Candida and candidias is (Caldero n e ,
R.A ., ed.), pp. 37-53, A SM Pres s , W a s h ington,
DC.
M uller, F .M .C., M . W eig, J. Peter and T. J. W als h
2000. A zole cros s -res is tance to ket o c onazole,
fluco n azole, itraconazole and voriconazole in
c linical Candida albicans is olates from HIVinfected children with oropharyngeal candidos is . J.
A ntimicrob. Chemother. 46: 323-342.
W h it e , T.C., 1997. Increas ed mRNA levels of
ERG16, CDR, and M DR1 correlate with increas es
in azole res is tance in Candi d a albicans is olates
f r o m a p a t ie n t in fe c t e d w i t h h u m a n
immunodeficiency virus . A ntimic ro b . A gents
Chemother., 41: 1482-1487.
W hite, T.C., M .A . P faller, M .G. Rinaldi, J. Smith,
and S.W . Redding, 1997. Stable azole drug
res is tance as s ociated with a s ubs train of Candi d a
albicans from an HIV-infected patient. Oral Dis . 3
(Suppl. 1): S102-S109.
M aric h a l, P., H. Vanden Bos s che, F.C. Odds , G.
Nobels , D.W . W arnock, V. Timmerman, C. Van
Broeckhoven, S. Fay and P. M os e-Lars en, 1997.
M olecular biolo gical characterization of an azoleres is tant Cand ida glabrata is olate. A ntimicrob.
A gents Chemother., 41: 2229-2237.
Van d e n Bos s che, H., P. M arichal, F.C. Odds , L.
Le Jeune and M .C. Coene, 1992. Characterization
of an azole-res is tant Candida glabrata is olate.
A ntimicrob. A gents Chemother., 36: 2602-2610.
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
7.
8.
9.
10.
11.
12.
13.
14.
15.
Kelly, S.L., D.C. Lamb and D.E. Kelly, 1999.
Y132H s ubs titution in Candida albi c a n s s terol 14
-demethylas e confers fluconazole res is tance by
prev e nting binding to haem. FEM S M icrobiol.
Lett., 180: 171-175.
Kelly, S.L., D.C. Lamb, J . Lo effler, H. Eins ele,
and D.E. Kelly, 1999. The G464S amino acid
s ubs titution in Candida albicans s terol 14 demethylas e caus es fluconazole res is tance in the
c linic through reduced affinity. Biochem. Biophy s .
Res . Commun., 262: 174-179.
Lamb, D.C., D.E. Kelly, T.C. W hite and S.L.
Kelly , 2000. The R467K amino acid s ubs titution in
Candida albicans s terol 14 -demethylas e caus es
d ru g res is tance through re d u c e d a ffin it y .
A ntimicrob. A gents Chemother., 44: 63-67.
M arichal, P ., L. Koymans , S. W illems ens , D.
Bellens , P. Verhas s elt, W . Luyten , M . Borgers , F.
C. Ramaekers , F.C. Odds and H. Va n d e n Bos s che,
1999. Contribut io n of mutations in the cytochrome
P450 14 -demethyla s e (Erg11p, Cyp51p) to azole
res is tance in Candida albicans. M icrobiology, 145:
2701-2713.
Pere a , S., J.L. Lopez-Ribot, W .R. Kirkpatrick, R.
K . M cA tee, R.A . Santillan, M . M artinez, D.
Calabres e, D. Sanglard a n d T.F. Patters on, 2001.
Prevalence of molecular mechanis ms of res is tance
to azole antifungal agents in Cand ida albicans
s trains dis playing high-level fluconazole res is tance
is olated from human immunodeficiency virus infected patients . A ntimicrob. A gents Chemother.,
45: 2676-2684.
Sanglard, D., F. Is cher, L. Koymans and J. Bille,
1998. A mino acid s ubs titutions in the cytochrome
P-450 lanos terol 14 -demethylas e (CYP51A 1) from
azo le-res is tant Candida albicans clinical is olates
contribute to res is tance to azole ant ifungal agents .
A ntimicrob. A gents Chemother., 42: 241-253.
A lberts on, G.D., M . Niimi, R.D . Cannon and H.F.
Jenkins on, 1996. M u ltiple efflux mechanis ms are
in v o lv e d in C a ndida albicans fluconazole
res is tance. A ntimicro b. A gents Chemother., 40:
2835-2841.
Lamb, D.C., D.E. Kelly, N.J. M anning and S.L.
Kelly, 1997. Reduced intracellular accumula tion of
azole antifungal res ults in res is tance in Candida
albicans is olate NCPF 3363. F EM S M icrobiol.
Lett., 147: 189-193.
M oran, G.P., D. Sanglard, S.M . Donn e lly, D.B.
Shanley, D.J. Sullivan and D.C. Coleman, 1998.
Id e ntification and expres s io n o f mu lt id ru g
trans porters res pons ible for flu conazole res is tance
in Candida dubliniensis. A ntimicro b. A gents
Chemother, 42: 1819-1830.
1333
16. Perea, S., J.L. Lopez-Ribot, B.L. W ickes , W .R.
Kirkpatric k, O .P . Dib, S.P. Bachmann, S.M .
Keller, M . Martinez and T.F. P a t ters on, 2002.
M olecular mechanis ms of fluconazole res is tance in
Candi d a d u b l i n i e nsis is olates from human
immunodeficiency virus -infecte d patients with
o ropharyngeal candidias is . A ntimicrob. A gents
Chemother, 46: 1695-1703.
17. Sanglard, D., K. Kuchler, F. Is cher, J.L. Paga n i,
M . M onod a n d J. Bille, 1995. M echanis ms of
res is tance to azole antifungal agents in Candida
albic ans is olates from A IDS patients involve
s pecific multidrug trans porters . A ntimicrob. A gents
Chemother, 39: 2378-2386.
18. Venkates warlu, K., D.W . Denning, N.J. M a nning,
and S.L. Kelly, 1995. Res is tance to fluconazole in
Can dida albicans from A IDS patients correlated
with reduced intracellular accu mu lation of drug.
FEM S M icrobiol. Lett., 131: 337-341.
19. Kelly, S.L., D.C. Lamb, A .J. Corran, B.C. Baldwin
and D.E. Kelly , 1995. M ode of action and
res is tance to azole antifungals as s ociated w ith the
formation of 14 -methylergos ta-8,24(28)-dien-3ß,6
-diol. Biochem. Biophys . Res . Commun., 207: 910915.
20. Kelly, S .L., D.C. Lamb, D.E. Kelly, J. Loeffler
a n d H. Eins ele, 1996. Res is tance to fluconazole
and amphotericin in Candida albicans from A IDS
patients . Lancet., 348: 1523-1524.
21. M iyazaki, Y., A . Geber, H. M iy a za ki, D. Falconer,
T. Parkins on, C. Hitchcock, B. Grimberg, K.
Nys waner, and J .E. Bennett, 1999. Cloning,
s equ e n c ing, expres s ion and allelic s equence
divers ity of ERG3 (C-5 s terol des aturas e gene) in
Candida albicans. Gene., 236: 43-51.
22. Pinjon, E., G.P. M oran, C.J. Jacks on, S.L. Kelly,
D . Sanglard, D.C. Coleman and D.J. Sulliv a n ,
2003. M olecular mechanis ms o f itraconazole
res is tance in Candida dubliniensis. A ntimicro b .
A gents Chemother, 47: 2424-2437.
23. W hite, A ., M .B. Goetz, 1994. A zole-res is tant
Candida albicans: report of two cas es of res is tance
to fluconazole and review. Clin Infe c t D is ., 19:
687-92.
24. Fan-Havard , P., D. Capano, S.M . Smith, 1991.
Development of res is t ance in Candida is olates
from patients receivin g prolonged antifungal
therapy.
A ntimicrob
A gents Chemother, 35:
2302-5.
25. M illon, L., A . M anteaux, G. Reboux, 1994.
Fluconazole-res is tant recurrent oral candidias is in
human immun o deficiency virus -pos itive patients :
p ers is tence of Candida albicans s trains with t h e
s ame genotype. J Clin M icrobiol., 32: 1115-8.
J. Appl. Sci. Res., 5(10): 1325-1334, 2009
26. Rautemaa, R., M . Richards on, M . Pfaller, KoukilaP. Kahkola, J. Perheentupa and H. Saxen, 2007.
Decreas ed s us ceptibility of Candida albicans to
azole antifungals : a complication of long-term
treatment in autoimmune polyendocrinopathycandidia s is -e c t o d e rma l d ys trophy (A PECED)
patients . J A ntimicr. Chemother, 60: 889-892.
27. Kirkpatrick, W .R., S .G. Revankar, R.K. M cA tee,
J.L. Lopez-Ribot, A .W . Fothergill, D.I. M cCarthy,,
S.E. Sanche, R.A . Cantu, M .G. Rinaldi and T.F.
Pat t ers on, 1998 J. Clin. M icrobiol., 36: 3007-3012.
28. Odds , F.C., L. Van Nuffel and G. Da ms , 1998. J
Clin. M icrobiol., 36: 2869-2873.
29. Pfaller, M .A ., D.J. Diekema, S.A . M es s e r, L.
Boyken, R.J. Hollis an d R.N . Jones , 2003.
A ntimicrob. A gents Chemother, 47: 1068-1071.
30. Pfaller, M .A ., D.J. Diekema, S.A . M es s er, R.J.
Hollis and R.N . Jones , 2004. Diagn. M icrobiol.
Infect. Dis ., 48: 101-105.
31. Pinjon, E., C.J. Jacks on, S .L. Kelly, D. Sanglard,
G. M oran, D.C. Coleman and D.J. Sullivan, 2005.
A ntimicrob. A gents Chemother, 49: 1312-1318.
32. M oran, G.P., D.J. Sullivan, M .C. H e nman, C.E.
M c Creay, B.J. Harrington, D.B. Shanley and D.C.
Coleman, 1997. A ntimicrob. A gents Chemother,
41: 617-623.
1334
33. Borg-Von Zepelin, M ., T. Niederhaus , U. Gros s ,
M . Seibold, M . M onod and K . Tintelnot, 2002.
A IDS., 16: 1237-1244.
34. A lberts on, G.D., M . Niimi, R.D. Cannon an d H .F .
Jenkins on, 1996. A ntimicrob. A gents Chemother,
40: 2835-2841.
35. Rex, J.H., T.J. W als h, J.D . Sobel, S.G. Filler, P.G.
Pappas , W .E. Dis mu ke s , 2000. Practice guidelines
for the treatment of candidias is . Infectious Dis eas es
Society of A merica. Clin Infect Dis ., 30: 662-78.
36. Vos s , A ., J.A . Kluytman s , J.G. Koeleman, L.
S p a njaard, C.M . Vandenbroucke-Grauls , H .A .
Verbrugh, 1996. O ccurrence of yeas t bloods tream
in fe ctions between 1987 and 1995 in five Dutc h
univers ity ho s pitals . Eur J Clin M icrobiol Infect
Dis ., 15: 909-12.
37. A bi-S a id, D., E. A nais s ie, O. Uzun, I. Raad, H.
P i n zc o w s ki, S . Va rt iv a ria n , 1 9 9 7 . T h e
epidemiology of hematogenous candidias is caus ed
by different Candida s pecies . Clin Infect Dis ., 24:
1122-8.
38. Hammer, K.A ., C.F. Cars on and T.V. Riley, 2000.
M e l aleuca alternifolia (tea tree) oil inhibits germ
tube formation by Candida albic ans. M edical
M ycology, 38: 355-62.