BioMe als 0: 1–7, 2004.
© 2004 Kluwe Academic Publishe s. P in ed in he Ne he lands. 1
Me al complexes o 1,10-phenan h oline-5,6-dione al e he suscep ibili y
o he yeas Candida albicans o Ampho e icin B and Miconazole
Ahmed Eshwika1, Ba y Coyle2, Michael De e eux3, Malachy McCann2& Ke in Ka anagh1,∗
1Medical Mycology Uni , NICB, Depa men o Biology, Na ional Uni e si y o I eland Maynoo h, Co. Kilda e,
I eland; 2Depa men o Chemis y, Na ional Uni e si y o I eland Maynoo h, Co. Kilda e, I eland; 3Dublin
Ins i u e o Technology, Ca hal B ugha S ., Dublin 1; ∗Au ho o co espondence (Tel: 353-1-708 3859, Fax:
353-1-708 3845, E-mail: ke in.ka [email protected].)
Recei ed 15 Oc obe 2003; Accep ed 28 No embe 2003
Abs ac
G ow h o he pa hogenic yeas Candida albicans in sub-MIC (minimum inhibi o y concen a ion) le els o
Cu(ClO4)2·6H2O and [Cu(phendio)3](ClO4)2·4H2O (phendio =1,10-phenan h oline-5,6-dione) inc eased he
concen a ion o miconazole and ampho e icin B equi ed o achie e he MIC90 whe eas p e-g ow h in AgClO4
and [Ag(phendio)2]ClO4 esul ed in a small dec ease in he ele an MIC90 alues. The coppe complexes educe
he oxygen consump ion o C. albicans while he sil e complexes inc ease oxygen consump ion. In addi ion, p e-
g ow h o cells in he coppe complexes esul ed in a lowe e gos e ol con en while he sil e complexes induced
an ele a ion in e gos e ol syn hesis.
The abili y o coppe and sil e complexes o al e he suscep ibili y o C. albicans o miconazole and ampho-
e icin B may be in luenced by hei ac ion on espi a ion, since educed espi a ion a es co ela e wi h educed
cellula e gos e ol which is he a ge o ampho e icin B. Lowe le els o e gos e ol ha e p e iously been asso-
cia ed wi h ele a ed ole ance o his d ug. In he case o educed sensi i i y o miconazole, ole ance may be
media ed by lowe e gos e ol syn hesis gi ing ise o ewe oxic side p oduc s once biosyn hesis is inhibi ed by
miconazole.
Abb e ia ions: phendio =1,10-phenan h oline-5,6-dione. phen =1,10-phenan h oline
In oduc ion
The yeas Candida albicans is esponsible o a ange
o supe icial and sys emic diseases in he immuno-
comp omised pa ien . Con en ional he apies o he
con ol o hese diseases ely upon he use o azole
and polyene d ugs which a ge e gos e ol biosyn-
hesis and al e memb ane pe meabili y, espec i ely
(Whi e e al., 1998, Abu Salah, 1996). In ecen yea s,
he appea ance o ungal s ains mani es ing esis ance
o con en ional d ugs (Canu o & Rode o, 2002) has
p omp ed he sea ch o no el an i- ungals wi h modes
o ac ion dis inc o he exis ing ange o an i- ungals.
Me al-based d ugs ep esen a no el g oup o an i-
ungal agen s wi h po en ial applica ions o he con-
ol o ungal in ec ions. P e ious wo k in ou labo -
a o ies has demons a ed ha in RPMI medium a
37oC he me al-based d ugs [Cu(phen)2(mal)]·2H2O,
[Mn(phen)2(mal)]·2H2O and [Ag(phen)2]ClO4(phen
=1,10-phenan h oline) inhibi he g ow h o C. al-
bicans by a ound 95% a a concen a ion o 5 µg/ml
(McCann e al. 2000; Coyle e al. 2003a). I was
es ablished ha bo h me al- ee phen and he me al-
phen complexes a ec mi ochond ial unc ion, e a d
he syn hesis o cy och omes b and c and uncouple es-
pi a ion. T ea men o ungal cells wi h he Cu(II) and
Ag(I) complexes esul ed in a educed amoun o e -
gos e ol in he cell memb ane and subsequen inc ease
in i s pe meabili y. Cells exposed o me al- ee phen
and he Cu(II) and Mn(II) complexes (bu no he Ag(I)
complex) demons a ed an ele a ion in oxygen up ake.
The gene al conclusion was ha he d ugs damage
mi ochond ial unc ion and uncouple espi a ion. Fu -
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AUTHOR’S PROOF!
2
he mo e, he ac ha he d ugs we e no uni o mly
ac i e sugges ed ha hei bioac i i y had a deg ee o
me al-ion dependency.
Mo e ecen ly, me al- ee phendio (phendio =
1,10-phenan h oline-5,6-dione) and he Ag(I) com-
plex [Ag(phendio)2]ClO4ha e been shown o cause
ex ensi e, non-speci ic DNA clea age o C. albicans,
dis up cell di ision and induce g oss dis o ions in
ungal cell mo phology (Coyle e al., 2003b). P e-
limina y expe imen s on cul u ed human cance cells
p oduced IC50 alues o 0.008 µg/ml (0.04 µM)
and 0.025 µg/ml (0.40 µM) o me al- ee phendio
and [Ag(phendio)2]ClO4, espec i ely (Coyle e al.
2003b). S udies by Igdalo e al. (1983) e ealed ha
bo h phendio and i s isome , 1,7-phenan h oline-5,6-
dione, inhibi he g ow h o S49 mouse lymphona cells
and S110 mouse cells, and al hough i was pos u-
la ed ha inhibi ion o DNA and RNA syn heses we e
majo componen s o he cy o oxic e ec s he phenan-
h olines we e p esumed o ha e mo e han one mode
o ac ion.
Me al-based d ugs ha e well es ablished ungi-
s a ic and ungicidal e ec s and he aim o he wo k
p esen ed he e was o e alua e he possibili y o using
Cu (II) and Ag (I) phendio complexes in combina-
ion wi h azole and polyene d ugs o he con ol o
C. albicans.
Ma e ials and me hods
Fungal isola e and cul u e condi ions
C. albicans MEN (a kind gi om D . Da id Ke idge,
Camb idge, UK) was g own o he s a iona y phase
(app oxima ely 1.5 ×108cells/ml) a 30 ◦C o e nigh
in YEPD b o h (2% (w/ ) glucose (Sigma Ald ich
Chemical Co., Do se , UK), 2% (w/ ) bac e iological
pep one (Sigma Ald ich) and 1% (w/ ) yeas ex ac
(Sigma Ald ich)) in an o bi al incuba o a 200 pm.
D ugs
Chemicals we e ob ained om comme cial sou ces
and used wi hou u he pu i ica ion. Cu(ClO4)2·6H2O
was pu chased om Sigma Ald ich and used wi hou
u he pu i ica ion. [Cu(phendio)3](ClO4)2·4H2O
and [Ag(phendio)2]ClO4we e syn hesized in acco d-
ance wi h he p ocedu es ou lined in McCann e al.
(2003).
An i-Candida suscep ibili y es ing o me al-based
d ugs
Solu ions o wa e -soluble coppe and sil e com-
plexes we e p epa ed by dissol ing 0.02 g o he solid
in s e ile dis illed wa e o yield a s ock solu ion o
200 µg/ml. The solu ions we e il e s e ilised using a
Millipo e memb ane il e (0.45 µm). S a iona y phase
cul u es o C. albicans we e ha es ed by cen i u-
ga ion (2220 ×g o 5 min in a Beckmann GS-6
cen i uge), washed wice wi h PBS and esuspended
a a inal densi y o 1 ×106cells/ml. Cell suspension
(100 µl) was added o each well o a 96-well mic o i e
pla e excep he i s column (con ol medium). Se ial
dilu ions o me al-based d ugs we e added o ows o
wells o cons uc a concen a ion g adien om 50–
0.78 µg/ml and he pla es we e incuba ed a 30 ◦C
o 24 h. The abso bance a 450 nm was de e mined
using a MRX spec opho ome e (Dynax Technology,
Chan illy, VA, USA) and he concen a ion ha was
capable o inhibi ing g ow h by 90% (MIC90) ela i e
o he con ol was calcula ed.
Ampho e icin B and miconazole suscep ibili y es ing
Yeas cul u es we e g own o he s a iona y phase
in an ibio ic medium 3 (AB 3, Oxoid) o e nigh a
30 ◦C and 200 pm, ha es ed by cen i uga ion and
dilu ed o 1 ×106/ml. Cells (1 ×105in 100 µl) we e
added o each ow o a 96-well pla e con aining am-
pho e icin B in se ial dilu ions (Sigma-Ald ich) om
2.5–0.0048 µg/ml in AB3 medium.
Miconazole suscep ibili y es ing was pe o med
using s a iona y phase C. albicans ha had been g own
in RPMI medium o e nigh (Sigma Ald ich). Cells
(100 µlo 1×106/ml) we e added o each ow o
a 96-well pla e con aining miconazole, se ially dilu ed
in RPMI medium om 20–0.19 µg/ml.
All pla es we e incuba ed a 30 ◦C o 24 h and he
op ical densi y was ead a 450 nm using a MRX spec-
opho ome e (Dynax Technology, Chan illy, VA,
USA).
Oxygen consump ion
Cells we e g own in YEPD b o h supplemen ed wi h
sub-MIC le els o sil e o coppe complexes a 30 ◦C
o 24 h, ha es ed by cen i uga ion and e-suspended
a a densi y o 1 ×108/ml in phospha e bu e ed saline
(PBS, pH 7.2). A Cla k Type oxygen elec ode (Ranks
B o he s, Camb idge, UK) was employed o de e m-
ine he espi a ion a e o cells. The a e o oxygen
B5271283. ex; 16/03/2004; 11:56; p.2
3
Fig 1. E ec o me al-based d ugs on he g ow h o C. albicans. Cells o C. albicans we e exposed o inc easing concen a ions o MBD and
he e ec on g ow h a e 24 h incuba ion was de e mined.
consump ion is exp essed as he numbe o µmoles o
oxygen consumed pe hi y sec pe 1 ×108cells.
S e ol ex ac ion and analysis
S e ols we e ex ac ed acco ding o he me hod o
A hing on–Skaggs e al. (1999). S a iona y phase
cells (1.3 g we weigh ) we e ha es ed and washed
wi h PBS (pH 7.2). Cells we e e-suspended in 20%
(w/ ) KOH and 60% ( / ) e hanol and placed in a
shaking wa e ba h (80–90 ◦C) o 1.5 h. Hep ane was
added o he solu ion which was hen agi a ed o
10 sec and he aqueous laye emo ed. The s e ol con-
en o he hexane laye was quan i ied using a dual
beam spec opho ome e o e he ange 250–300 nm.
S a is ical analysis
The K uskal–Wallis es was pe o med, whe e app o-
p ia e, on all esul s using he SigmaS a S a is ical
Analysis Sys em (Ve sion 1.00). Values a e p esen ed
as ±SE o he mean o da a om h ee independen
expe imen s.
Resul s
The e ec o selec ed Cu(II) and Ag(I) phendio
complexes on he g ow h o C. albicans was as-
sessed. The esul s indica e (Figu e 1) ha AgClO4
and [Ag(phendio)2]ClO4display MIC90 alues o ap-
p oxima ely 6.0 µg/ml while Cu(ClO4)2·6H2Oand
[Cu(phendio)3](ClO4)2·4H2OdisplayMIC
90 alues
o >50 and 46 µg/ml, espec i ely. In all sub-
sequen assays, concen a ions co esponding o 1/4
MIC90 alues o hese complexes alues we e em-
ployed i.e. AgClO4and [Ag(phendio)2]ClO4a a
concen a ion o 1.5 µg/ml and Cu(ClO4)2·6H2O
and [Cu(phendio)3](ClO4)2·4H2O a concen a ions
o 12.5 µg/ml and 11.5 µg/ml, espec i ely. Sub-MIC
alues we e employed in all subsequen assays so ha
any e ec on d ug suscep ibili y would no be due o
he ungis a ic e ec s o hese compounds
Expe imen s we e pe o med o de e mine whe he
p e-g ow h o C. albicans in he p esence o di -
e en me al complexes a ec ed he subsequen sus-
cep ibili y o cells o azole and/o polyene d ugs.
Cells we e p e-g own in medium supplemen ed wi h
1/4 MIC90 o he Cu(II) and Ag(I) phendio com-
plexes o 24 h a 30 ◦C and ha es ed by cen i-
uga ion. An i- ungal suscep ibili y assays we e pe -
o med as desc ibed. The esul s (Figu e 2) in-
dica e ha p e-g ow h o C. albicans in medium
supplemen ed wi h 12.5 µg/ml Cu(ClO4)2·6H2O
o 11.5 µg/ml [Cu(phendio)3](ClO4)2·4H2O signi-
ican ly dec eases he suscep ibili y o C. albic-
ans o miconazole (p <0.001) (con ol MIC90 =
7.94 ±1.2 µg/ml, cells p e-g own in 1/4 MIC
Cu(ClO4)2·6H2OMIC
90 =10.9 ±0.2 µg/ml, cells
p e-g own in 1/4 MIC [Cu(phendio)3](ClO4)2·4H2O
MIC90 =11.4±0.3 µg/ml ). In he case o he sil-
e complexes, p e-g ow h o C. albicans in 1.5 µg/ml
AgClO4o [Ag(phendio)2]ClO4inc eases he suscep -
ibili y o he cells o miconazole. Cells p e-g own
in 1/4 MIC90 AgClO4show a miconazole MIC90
alue o 3.24 ±0.09 µg/ml while hose p e-g own in
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4
Fig 2. Suscep ibili ies o Miconazole o C. albicans p e-g own in he p esence o sub-MIC90 le els o me al complexes. The suscep ibili y o
C. albicans o miconazole was de e mined a e cells had been i s p e-g own in sub- MIC90 le els o me al-based d ug.
1/4 MIC90 [Ag(phendio)2]ClO4demons a e a MIC90
alue o 4.95 ±0.27 µg/ml.
The suscep ibili y o C. albicans o he polyene
d ug ampho e icin B was also a ec ed ollowing
p e-g ow h o he cells in medium supplemen ed
wi h coppe o sil e complexes a 1/4 MIC90 al-
ues (Figu e 3). In he case o p e-g ow h in 1/4
MIC90 Cu(ClO4)2·6H2O he amoun o ampho e i-
cin B equi ed o achie e he MIC90 inc eases o
0.029 ±0.007 µg/ml om 0.024 ±0.003 µg/ml
(con ol) and ollowing p e-g ow h in a simila con-
cen a ion o [Cu(phendio)3](ClO4)2·4H2O inc eases
o 0.03 ±0.003 µg/ml (p =0.238). In he case
o p e-exposu e o he sil e complexes he e is a
dec ease in he amoun o ampho e icin B equi ed
o achie e he MIC90. P e-g ow h o C. albicans in
ei he o he sil e complexes educes he amoun
o ampho e icin B equi ed o achie e he MIC90
om 0.024 ±0.003 µg/ml o 0.019 ±0.001 µg/ml
(p =0.238) (Figu e 3).
Azoles, such as miconazole, inhibi he ac ion
o lanos e ol 14α-deme hylase which con ols an es-
sen ial in e media e s ep in he biosyn hesis o e -
gos e ol (Whi e e al., 1998, Daum e al., 1998).
In con as , polyenes, such as ampho e icin B, bind
o e gos e ol in he ungal cell memb ane c ea ing
po es h ough which cell cons i uen s may escape
(Abu Salah, 1996). E gos e ol biosyn hesis is an oxy-
gen dependen p ocess and also equi es NADPH
which is syn hesised in he ae obically espi ing mi-
ochond ion. Consequen ly, expe imen s we e pe -
o med o es ablish whe he he al e ed suscep ib-
ili y o C. albicans o miconazole and ampho e i-
cin B was due o he abili y o he coppe o sil-
e complexes o al e espi a ion. The esul s (Fig-
u e 4) demons a e ha p e-g ow h o cells in 1/4
MIC90 o he coppe complexes (Cu(ClO4)2·6H2O
and [Cu(phendio)3](ClO4)2·4H2O) esul s in a educ-
ion in he cellula espi a ion a e (p <0.001) whe eas
g ow h o cells in he p esence o he sil e com-
plexes (AgClO4and [Ag(phendio)2]ClO4)inc eases
he espi a ion a e o C. albicans (p <0.001).
The e gos e ol biosyn he ic pa hway is he a -
ge o he azole d ugs (Whi e e al. 1998) whils
polyenes bind e gos e ol in he cell memb ane and
c ea e po es (Abu Salah, 1996). Quan i ica ion o
e gos e ol in cells g own in medium supplemen ed
wi h sub MIC90 concen a ions o he me al-based
d ugs demons a ed ha g ow h in he p esence o
coppe complexes esul ed in a educed e gos e ol
con en , while g ow h in medium supplemen ed wi h
he sil e complexes esul ed in an inc ease in cel-
lula e gos e ol (Figu e 5). In e es ingly, hose com-
pounds ha inc ease he espi a ion a e (AgClO4and
[Ag(phendio)2]ClO4) also cause an inc ease in e -
gos e ol con en while hose ha dep ess espi a ion
(Cu(ClO4)2·6H2O and [Cu(phendio)3](ClO4)2·4H2O)
lead o a educ ion in e gos e ol con en .
B5271283. ex; 16/03/2004; 11:56; p.4
5
Fig 3. Suscep ibili ies o Ampho e icin B o C. albicans p e-g own in he p esence o sub-MIC90 le els o me al complexes. The suscep ibili y
o C. albicans o ampho e icin B was de e mined a e cells had been i s p e-g own in sub- MIC90 le els o me al-based d ug.
Fig 4. Respi a ion a es o C. albicans cells p e-g own in sub-MIC90 le els o me al complexes. The oxygen consump ion a es o C. albicans
cell p e-g own in sub-MIC90 le els o me al-based d ug we e de e mined using a Rank Oxygen elec ode and exp essed as µmoles o oxygen
consumed pe 108cells pe 30 sec.
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6
NEW ARTWORK PLEASE!
Fig 5. E gos e ol p o iles o C. albicans cells p e-g own in
sub-MIC90 le els o me al complexes. (Rep esen a i e igu e). The
ela i e amoun s o e gos e ol in cells p e- ea ed wi h sub-MIC90
concen a ions o me al-based d ug we e asce ained spec opho o-
me ically o e he ange 250–300 nm.
Discussion
Me al-based d ugs ep esen a no el g oup o an i-
mic obial agen s wi h po en ial he apeu ic applica-
ions (Coyle e al. 2003a; McCann e al. 2000). Wi h
he ad en o ungal isola es mani es ing esis ance o
azole and polyene d ugs (Canu o & Rode o, 2002)
he e is a equi emen o new d ugs wi h al e na i e
modes o ac ion o wi h he abili y o inc ease he
e icacy o exis ing p esc ip ion d ugs. Many me al-
based d ugs display modes o ac ion dis inc o hose
o he p esc ip ion an i- ungals (Coyle e al. 2003a)
possibly allowing hei use whe e esis ance o con-
en ional d ugs has eme ged (Whi e e al. 1998). In
addi ion, hei di e en mode(s) o ac ion may be u il-
ised by employing such d ugs in conjunc ion wi h
exis ing d ugs in o de o a ge wo (o mo e) si es
wi hin he ungal cell and hus aising he possibili y
o achie ing he same he apeu ic e ec by educing
he equi ed amoun o an azole o polyene d ug.
Con en ional an i- ungal d ugs such as polyenes o
azoles a ge e gos e ol in he cell memb ane o he
e gos e ol biosyn he ic pa hway, espec i ely. In he
wo k p esen ed he e we ha e e alua ed he abili y o
Cu(II) and Ag(I) phendio complexes o al e he sus-
cep ibili y o C. albicans o con en ional azole and
polyene d ugs.
The coppe and sil e complexes e alua ed he e
demons a ed ungis a ic p ope ies bu o his wo k
sub-MIC90 alues we e employed o asce ain hei
e ec on he sensi i i y o C. albicans o he con en-
ional an i ungal d ugs. Sub-MIC le els we e chosen
o ensu e ha he obse ed e ec was no due o he
ungis a ic ac ions o he me al complex. When cells
a e p e-g own in sub-MIC90 le els o he sil e o
coppe complexes and hen assessed o hei esponse
o miconazole o ampho e icin B he e is e idence o
al e a ions in suscep ibili y. P e-g ow h in 1/4 MIC90
Cu(ClO4)2·6H2O o [Cu(phendio)3](ClO4)·4H2Oin-
c eased he ole ance o C. albicans o miconazole
and ampho e icin B, whe eas p e-g ow h in equi alen
concen a ions o AgClO4and [Ag(phendio)2ClO4]
lowe s he suscep ibili y o C. albicans o miconazole
and ampho e icin B.
The al e a ion in suscep ibili y o miconazole and
ampho e icin B may occu due o he ac ha
he me al complexes which inhibi espi a ion (i.e.
Cu(ClO4)2·6H2O and [Cu(phendio)3](ClO4)2·4H2O)
also cause a educ ion in he cellula e gos e ol con-
en . Azoles, such as miconazole, a ge lanos e ol
14α-deme hylase which egula es an in e media e s ep
in e gos e ol biosyn hesis (Mo schhause , 2002). The
azoles can kill cells by inhibi ing his enzyme which
subsequen ly leads o a dec ease in e gos e ol con en
o he ungal cell memb ane. In addi ion, he inhibi ion
o he ac ion o 5,6-s e ol desa u ase by luconazole
leads o he accumula ion o oxic in e media es (such
as 14α-me hyl ecos e ol) which may p o e a al o he
ungal cell (Gebe e al. 1995). The al e ed suscep ib-
ili y o miconazole may be due o educed e gos e ol
biosyn hesis esul ing in ewe oxic side p oduc s be-
ing o med once he ac ion o he deme hylase has
been inhibi ed by miconazole. Enhanced e gos e ol
biosyn hesis may inad e en ly lead o g ea e p oduc-
ion o oxic side-p oduc s once he azole-media ed
inhibi ion o s e ol biosyn hesis occu s.
Reduced le els o e gos e ol in he ungal cell
memb ane p o ide ewe binding si es o ampho e i-
cin B. Consequen ly a highe concen a ion o ampho-
e icin B is equi ed o e a d he g ow h o he cell.
Reduc ions in s e ol le els in C. albicans ha e been
iden i ied p e iously as a mechanism o inc eased
g ow h in he p esence o ampho e icin B (Kelly e al.
1997; Whi e e al. 1998). Dis up ions o he genes in
he e gos e ol biosyn hesis pa hway cause a dec ease
in e gos e ol in C. glab a a andaninc easeind ug
ole ance, pa icula ly o ampho e icin B (Gebe e al.
1995; Vazquez e al. 1996). Inhibi ion o espi a ion
in C. albicans by e y h omycin leads o a d op in e -
gos e ol and a concomi an inc ease in ole ance o
ampho e icin B (Ge agh y & Ka anagh, 2003a). In
addi ion, dis up ion o mi ochond ial unc ion leads o
B5271283. ex; 16/03/2004; 11:56; p.6
7
ole ance o ampho e icin B due o deple ed e gos e ol
syn hesis (Ge agh y & Ka anagh, 2003b).
The equi emen o a unc ional mi ochond ion
in e gos e ol biosyn hesis is well cha ac e ised and
a ises om he p o ision o NADPH o squalene di-
me isa ion (Pa ks & Casey, 1995). In addi ion, E g1
encodes squalene epoxidase, which con e s squalene
o 2,3-oxidosqualene. This is an oxygen-dependen
s ep, and in a cell wi h educed espi a ion he e would
a consequen educ ion in he syn hesis o e gos e ol
(Daum e al. 1998), hus leading o he educed e -
gos e ol con en e iden in cells exposed o he coppe
complexes.
An inc ease in espi a ion, as e iden when
cells a e p e-g own in he p esence o AgClO4and
[Ag(phendio)2]ClO4s imula es e gos e ol p oduc ion.
Thus, he e is mo e e gos e ol o ampho e icin B o
bind and, as a consequence, lowe ing he amoun o
he polyene necessa y o inhibi ion o cell g ow h. In
he case o he al e ed sensi i i y o miconazole, he
sil e complexes s imula e espi a ion and e gos e ol
biosyn hesis and consequen ly may indi ec ly lead o
he gene a ion o mo e oxic side-p oduc s once he
cells a e exposed o miconazole.
The wo k p esen ed he e demons a es ha using
sub-MIC le els o coppe o sil e dione complexes i
is possible o al e he amoun o an azole o polyene
d ug equi ed o inhibi he g ow h o C. albicans.This
e ec appea s o be media ed h ough al e a ions in
he espi a ion a e o he cell, he eby in luencing he
amoun o e gos e ol syn hesis. This opens he possib-
ili y o u ilising, non- oxic le els o sil e complexes
o s imula e espi a ion and e gos e ol p oduc ion wi h
a concomi an educ ion in he amoun o miconazole
o ampho e icin B equi ed o achie e he ele an
MIC90.
Acknowledgemen
This wo k was suppo ed by a g an om he Depa -
men o Educa ion, Libya.
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