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ARTICLE IN PRESS ModelICRES-25569; No. of Pages 9Microbiological
Research xxx (2013) xxx– xxx
Contents lists available at SciVerse ScienceDirect
Microbiological Research
jo ur nal ho me p age: www.elsev ier .com/ locate /micres
ntifungal activity of gemini quaternary ammonium salts
wa Obłąka,∗, Agata Piecucha, Anna Krasowskab, Jacek Łuczyński
c
Institute of Genetics and Microbiology, University of Wroclaw,
Przybyszewskiego 63/77, 51-148 Wroclaw, PolandFaculty of
Biotechnology, University of Wroclaw, Przybyszewskiego 63/77,
51-148 Wroclaw, PolandFaculty of Chemistry, Wroclaw University of
Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wroclaw, Poland
a r t i c l e i n f o
rticle history:eceived 3 February 2013eceived in revised form 28
May 2013ccepted 1 June 2013
a b s t r a c t
A series of gemini quaternary ammonium chlorides and bromides
with various alkyl chain and spacerlengths was synthesized. The
most active compounds against fungi were chlorides with 10 carbon
atomswithin the hydrophobic chain. Among these compounds were few
with no hemolytic activity at minimal
vailable online xxx
eywords:accharomyces cerevisiaeandia albicansemini quaternary
ammonium salts
inhibitory concentrations. None of the tested compounds were
cytotoxic and mutagenic. Cationic geminisurfactants poorly reduced
the adhesion of microorganisms to the polystyrene plate, but
inhibited thefilamentation of Candida albicans. One of the tested
compounds eradicated C. albicans and Rodotorulamucilaginosa
biofilm, what could be important in overcoming catheter-associated
infections. It was alsoshown that gemini surfactants enhanced the
sensitivity of C. albicans to azoles and polyenes, thus theymight
be potentially used in combined therapy against fungi.
. Introduction
Surfactants as surface-active compounds can interact with
theellular membranes of microorganisms and in consequence be
goodntimicrobial agents (Shirai et al., 2006, 2009; Hoque et al.,
2012;renier et al., 2012). On the other hand surfactants easily
absorbt liquid/solid interphases, coat surfaces and protect them
fromdhesion of microorganisms (McCarron et al., 2007).
The diseases caused by fungal colonization have become a
bigroblem due to ever growing strain resistance (Pfaller, 2012).
Forhis reason the antifungal activity of many known and commonlysed
antifungals such as fluconazole is still decreasing and the
solu-ion to this problem could be finding new active compounds
andorking out new strategies of extermination of pathogenic
fungi.
Candida albicans is the most well known opportunistic
pathogenhich lives in yeast or filamentous form. C. albicans
biofilms are
he most difficult form to eradicate due to e.g., a polymeric
matrix,hich strongly protects mycelium against penetration by
drugs
Tournu and Van Dijck, 2012). Infections, like fungemia or
meningi-is caused by Rhodotorula sp. are less common, however they
mightccur if the patient is immunosupressed. A saprophytic form
ofhis fungus has been collected from skin, vaginal and
respiratory
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racts, but a colonizing form has been found in catheters,
contactenses, bronchoscopes and hemodialysis machines, where it
canreate biofilms (Savini et al. 2008). Thus adhesion, the first
step to
Abbreviation: Gemini-QAS (bis-QAS), gemini quaternary ammonium
salt.∗ Corresponding author. Tel.: +48 883328686.
E-mail address: [email protected] (E. Obłąk).
944-5013/$ – see front matter © 2013 Published by Elsevier
GmbH.ttp://dx.doi.org/10.1016/j.micres.2013.06.001
© 2013 Published by Elsevier GmbH.
biofilm formation seems to be a good moment to apply
antifungalcompounds. Surfactants are perfect compounds for
diminishing thebiofilm creation risk.
Gemini surfactants are a class of amphiphilic compounds
builtfrom two classic surfactant moieties (of two hydrophobic
tailsand two cationic head groups per molecule) bound together by
aspacer group. In comparison with corresponding single chain
sur-factants (mono-QASs), these surfactants (gemini-QASs, also
calledbis-QASs) are more efficient in lowering surface tension and
havemuch lower critical micelle concentration (CMC) (Lachowicz et
al.,1992, 1995; Fuhrhop and Wang, 2004). Due to their higher
surfaceactivity they have excellent dispersion stabilization and
soil clean-up properties (Conte et al., 2005). These compounds are
widely usedas effective emulsifiers and dispersing agents (Schnell
et al., 2008).Moreover, they appear to be excellent for creating
complexes withDNA and are effective in mediating transfection. Due
to their con-struction, DNA carrier molecules built from gemini
surfactants areable to deliver genes to cells of almost any DNA
molecule size (McGregor et al., 2001; Pullmannova et al., 2012; Kim
et al., 2011).
Single tail single head cationic surfactants show good
antimicro-bial activity, however they exhibit hemolytic activity
(Shalel et al.,2001; Vieria and Carmona-Riberio, 2006). Amino acid
based surfac-tants from the cationic guanidine group have strong
antimicrobialactivity and are less toxic to human cells and more
environmen-tally friendly (Moran et al., 2004). Cationic gemini
surfactantsbased on arginine have a very low critical micelle
concentration
y of gemini quaternary ammonium salts. Microbiol Res (2013),
(CMC) and high antimicrobial activity, but are toxic for human
cells(Perez et al., 2002; Castillo et al., 2004). Colomer and
co-workers(2011) synthesized a series of gemini, lysine-based
surfactants andtested their antimicrobial and hemolytic activity.
Generally the
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ARTICLE ING ModelMICRES-25569; No. of Pages 92 E. Obłąk et al.
/ Microbiological Re
N
H3C
H3C
CH2 COO CnH2n+1
(CH2)x
N CH2H3C
H3C
COO CnH2n+1
2 Y
x = 2, Y = Cl (TMEG-n Cl)
x = 2, Y = Br (TM EG-n Br)x = 3, Y = Cl (TM PG-n C l)x = 3, Y =
Br (TMPG-n Br)
n = 6, 8, 10, 12, 14
Fig. 1. Structure of tested gemini quaternary ammonium salts,
derivativesof N,N,N′ ,N′-tetramethylethylenediamine (TMEG-n Cl:
N,N′-Bis[2-(n-alkyloxy)-2-oxoethyl]-N,N,N′ ,N′-tetramethylethylene-
diammonium dichlorides or TMEG-n
Br:N,N′-Bis[2-(n-alkyloxy)-2-oxoethyl]- N,N,N′
,N′-tetramethylethylenediammoniumdibromides) or N,N,N′
,N′-tetramethyl-1,3-propanediamine (TMPG-n Cl:
N,N′-Bis[2-(n-alkyloxy)-2-oxoethyl]-N,N,N′
,N′-tetramethyl-1,3-propanediammonium dichlo-rides or TMPG-n Br:
N,N′-Bis[2-(n-alkyloxy)-2-oxoethyl]-N,N,N′ ,N′-tetramethyl-1
abl
otigC
2
2
1(ddh(
2
sc(N1a
2
o1mtwrawfho
In the present study, the following fungal strains were
used:
,3-propanediammonium dibromides).
ntibacterial activity of lysine derivatives was lower than
arginine-ased compounds. Moreover, single-chain lysine surfactants
were
ess hemolytic than corresponding gemini amphiphiles.In this work
we describe the biological activity of the series
f gemini quaternary ammonium salts having betaine based esterype
alkyl chain arrangements – their hemolytic activity, cytotox-city
and mutagenic potential as well as their activity against therowth,
adhesion and biofilm formation of two pathogenic fungi –andida
albicans and Rhodotorula mucilaginosa.
. Materials and methods
.1. Materials
Chloroacetyl chloride (98%), bromoacetyl bromide (≥98%),-hexanol
(98%), 1-octanol (≥99%), 1-decanol (99%), 1-dodecanol98%),
1-tetradecanol (97%), N,N,N′,N′-tetramethylethylene-iamine (99%),
N,N,N′,N′-tetramethyl-1,3-propanediamine (99%),ichloromethane (pure
p.a.), acetonitrile (pure p.a.), sodiumydrogen carbonate (pure
p.a.) and magnesium sulfate anhydrouspure p.a.) were all purchased
from Sigma–Aldrich.
.2. Synthesis of gemini quaternary ammonium salts
A series of cationic gemini surfactants was synthesized by a
two-tep procedure, as reported before (Tehrani-Bagha et al.,
2012),onstituting the appropriate n-alkyl �-chloro- or
�-bromoacetatesABr/ClAs) synthesis and in the next
step–quaternization of,N,N′,N′-tetramethylethylenediamine or
N,N,N′,N′-tetramethyl-,3-propanediamine with ABr/ClAs (for the
structure see Fig. 1,bbreviated as TMEG-n Br/Cl or TMPG-n Br/Cl; n
= 6, 8, 10, 12, 14).
.3. Synthesis of n-alkyl-˛-halo-acetates (general procedure)
n-Alkyl �-bromo/chloro-acetates were synthesized in a reactionf
n-alkanoles (1-hexanol, 1-octanol, 1-decanol, 1-dodecaconol
or-tetradecanol) with chloroacetyl chloride (or bromoacetyl
bro-ide) in dichloromethane as a solvent. Thus, 0.5 mol of the
respec-
ive primary alcohol, dissolved in 400 cm3 of dichloromethane,as
stirred under reflux and 0.7 mol of chloroacetyl chlo-
ide/bromoacetyl bromide in 100 cm3 of dichloromethane wasdded
stepwise, while the hydrobromide/hydrochloride formedas trapped in
a NaOH solution. The reaction mixture was refluxed
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or an additional 8 h, then cooled and neutralized with
sodiumydrocarbonate, and washed with water several times and
driedver anhydrous magnesium sulphate.
PRESSsearch xxx (2013) xxx– xxx
2.4. Quaternization of tetramethyl-diamines
withn-alkyl-˛-halo-acetates (general procedure)
After the solvent evaporation, n-alkyl-�-halo-acetates
wereachieved in an 80% yield and immediately used for the
quaterniza-tion step of N,N,N′,N′-tetramethylethylenediamine or
N,N,N′,N′-tetramethyl-1,3-propanediamine. In the latter step 0.1
mol of thediamine in acetonitrile was heated at 80 ◦C and 0.2 mol
of a givenalkyl �-bromoacetate (or �-chloroacetate) also in
acetonitrile wasadded drop wise to the reaction mixture. The
mixture was refluxedfurther for 30 h at 80 ◦C, then cooled in a
refrigerator and the precip-itated product was filtered off (yield
25–40%). The crude productswere recrystallized from a
hexane/chlorophorm solvent systemwith (v/v) ratio dependent on the
compound and purity of the prod-uct. The best effect of the process
was obtained using procedure asfollow. Crude product was dissolved
in appropriate volume of chlo-roform under reflux and then n-hexane
was added drop wise untilthe beginning of precipitation was
observed. Solution was cooled tothe room temperature and kept
several days in refrigerator. Crys-tallized product was filtered
off, dried and its purity was checked(sharp m.p., 1H NMR spectra).
If the purity was not satisfying crys-tallization was repeated. The
scheme of 2-step synthesis of geminisurfactants is shown in Fig.
2.
The surfactants were purified by repeated crystallization
untilno impurities could be detected by NMR spectra. The chemi-cal
structure of compounds was determined by their 1H NMR(Brucker
Avance 300 MHz, CDCl3, internal standard TMS, ı ppm).The results
indicate that the gemini quaternary ammonium bro-mides/chlorides
are at least 98 mole% pure, as was confirmed by thevery narrow
range of melting points. The 1H NMR spectra (shownas examples) are
described as follows:
TMEG-10Cl: 0.86 [6H, t, J = 5.7 Hz, 2(–CH3)]; 1.25–1.40 [28H,
m,2((–CH2)7–CH3)]; 1.66–1.79 [4H, m, 2(O–CH2–CH2)]; 3.774 [12H,
s,2(–N–(CH3)2)]; 4.17 [4H, t, J = 6.9 Hz, (–N–CH2–CH2–N)]; 4.69
[4H,s, 2(–N–CH2–COO–)]; 4.83–4.93 [4H, m, 2(O–CH2)];
TMEG-12Cl: 0.86 [6H, t, J = 6.6 Hz, 2(–CH3)]; 1.24–1.41 [36H,
m,2((–CH2)9–CH3)]; 1.66–1.80 [4H, m, 2(O–CH2–CH2–)]; 3.63 [12H,s,
2(–N–(CH3)2)]; 4.17 [4H, t, J = 6.9 Hz, (N–CH2–CH2–N)]; 4.73 [4H,s,
2(–N–CH2–COO–)]; 4.84–4.92 [4H, m, 2(O–CH2)];
TMEG-14Cl: 0.86 [6H, t, J = 6.6 Hz, 2(–CH3)]; 1.24–1. 37 [44H,
m,2((–CH2)11–CH3)]; 1.66–1.79 [4H, m, 2(O–CH2–CH2–)]; 3.54 [12H,s,
2(–N–(CH3)2)]; 4.18 [4H, t, J = 6.9 Hz, (N–CH2–CH2–N)]; 4.48 [4H,s,
2(–N–CH2–COO)]; 4.89–4.99 [4H, m, 2(O–CH2)];
TMPG-10Cl: 0.85 [6H, t, J = 6.6 Hz, 2(–CH3)]; 1.24–1.48 [28H,
m,2((–CH2–(CH2)7–CH3)]; 1.61–1.65 [4H, m, 2(–O–CH2–CH2)];2.68–2.89
(2H, m, (N–CH2–CH2–CH2–N)); 3.63 [12H, s,2(–N–CH3)2)]; 3.97 (4H, t,
J = 6.9 Hz, 2(–N–CH2–CH2–CH2–N–);4.15 (4H, s, 2(–N–CH2–COO–));
4.83–4.99 [4H, m, 2(O–CH2)];
TMPG-12Cl: 0.86 (6H, t, J = 6.6 Hz, 2(–CH3)); 1.24–1.49 [36H,
m,2((–CH2–(CH2)9–CH3)]; 1.61–1.66 [4H, m, 2(–O–CH2–CH2)];2.71–2.81
(2H, m, (N–CH2–CH2–CH2–N)); 3.64 [12H, s,2(–N–CH3)2)]; 3.99 (4H, t,
J = 6.9 Hz, 2(–N–CH2–CH2–CH2–N–);4.15 (4H, s, 2(–N–CH2–COO–));
4.84–4.93 [4H, m, 2(O–CH2)];
TMPG-14Cl: 0.86 (6H, t, J = 6.6 Hz, 2(–CH3)); 1.24–1.41 [44H,
m,2((–CH2–(CH2)11–CH3)]; 1.62–1.86 [4H, m, 2(–O–CH2–CH2)];2.59–2.78
(2H, m, (N–CH2–CH2–CH2–N)); 3.57 [12H, s,2(–N–CH3)2)]; 3.86 (4H, t,
J = 6.9 Hz, 2(–N–CH2–CH2–CH2–N–);4.15 (4H, s, 2(–N–CH2–COO–));
4.76–4.91 [4H, m, 2(O–CH2)]
2.5. Strains
y of gemini quaternary ammonium salts. Microbiol Res (2013),
Candida albicans ATCC 90028, Candida parapsilosis IHEM
3270,Rhodotorula mucilaginosa IHEM 18459 and Saccharomyces
cerevisiae�1278b.
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ARTICLE IN PRESSG ModelMICRES-25569; No. of Pages 9E. Obłąk et
al. / Microbiological Research xxx (2013) xxx– xxx 3
salts
2
mmbPewB(c
2
uoiif
2
FoPcftT
2
cpcaDactopnt
Fig. 2. Synthetic rout for the synthesis of gemini quaternary
ammonium
.6. Minimal inhibitory concentration (MIC)
To establish the antifungal activity of the tested
compounds,inimal inhibitory concentration (MIC) on 96-well
polystyreneicrotiter plates (Sarstedt) was determined. Strains were
incu-
ated with compounds in the range of concentrations 10–800
�M.lates were incubated for 48 h at 28 ◦C in YPG medium (1%
yeastxtract Difco, 1% peptone Difco, 2% glucose) and optical
densityas measured using a microplate reader at A590nm (ASYS UVM
340iogenet) according to Clinical and Laboratory Standards
Institute2008), 3rd ed. M27-A3. Negative and growth control wells
did notontain surfactants.
.7. Cytotoxicity assay
For cytotoxicity measurements alamarBlue Assay (Sigma) wassed.
The S. cerevisiae �1278b strain was incubated for 12 h withr
without a given compound. AlamarBlue (resazurin) was addedn an
amount equal to 10% of the volume in the well. The plate
wasncubated for 4 h in the dark. Conversion of resazurine to a
reducedorm (pink color) was observed.
.8. Hemolysis
Gemini-QAS were tested for hemolytic activity, as described
byalkinham III et al., 2012. 5 mL of sheep blood was centrifuged
tobtain morphotic elements (2500 rpm, 15 min), washed 3 times inBS
(pH 7.4) and resuspended in PBS. The compound at various
con-entrations was mixed with 100 �L of erythrocytes and
incubatedor 1.5 h at 37 ◦C. Absorbance was measured at � = 540 nm.
As posi-ive and negative controls, PBS and 1% SDS (respectively)
were used.his test was repeated at least three times.
.9. Ames’ test
Two strains of Salmonella Typhimurium, TA98 and TA100, defi-ient
in the synthesis of histidine, were used according to methodroposed
by Ames et al., 1975. The tested compound at givenoncentration and
100 �L of bacterial culture (108 cell/mL) weredded to 2 mL of top
agar and spread on the plate with minimalavis medium. The mixture
without tested compound was useds negative control. Plates were
incubated for 48 h at 37 ◦C andolonies were counted. As positive
control cisplatin was used inhe concentration 5 �g per plate. The
mutagenic ratio (MR) – ratio
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f the number of revertants grown in the presence of tested
com-ound to the number of spontaneously appeared revertants (on
theegative control) was calculated. Mutagenic ratio equal or
higherhan 2.0 determines the mutagenic potential of tested
compound.
. Y–chlorine or bromine; n = 6, 8, 10, 12 or 14; x = spacer
length (2 or 3).
Mutagenic ratio lower than 1.7 indicates lack of mutagenic
activ-ity. Values of MR between 1.7 and 2.0 designate the compound
aspotential mutagen. The test was repeated three times with
similarresults.
2.10. The influence of gemini surfactants on Candida
albicanssensitivity to azoles and polyenes
To determine the influence of the tested gemini-QAS on
C.albicans sensitivity to antifungals (azoles and polyenes), C.
albicanswere diluted in YPG on microtiter plates. Gemini cationic
sur-factants at final concentrations of 1/2 MIC, and itraconazole
andfluconazole at final concentrations of 5 and 150 �g/mL,
respec-tively, were applied. From the polyene group 0.3 �M
amphotericinB was used. Compounds were added separately or in
combinations:gemini surfactant-azoles or gemini
surfactants-amphotericin B.Cells were incubated at 28 ◦C for 24 h
and viability was measuredusing a microplate reader at A590nm (ASYS
UVM 340 Biogenet). Neg-ative and growth control wells did not
contain surfactants. This testwas repeated at least three
times.
2.11. Effect of gemini quaternary ammonium salts on theadhesion
and biofilm removal
C. albicans and R. mucilaginosa were grown on a YPG medium at30
◦C for 24 h. The cells were then washed once with
phosphate-buffered saline (PBS, pH 7.4) and diluted in fresh YPG
medium toa final concentration of 5 × 106 cells/mL. Gemini
surfactants wereadded to a 96-well flat-bottom polystyrene plate
(Sarstedt) at con-centrations: 10–240 �M, and the plate was
incubated for 2 h at37 ◦C with shacking. Then, the plate was washed
with distilledwater and the tested strains were added to the final
culture vol-ume, 100 �L in every well. The plate was incubated at
37 ◦C for 2 hto induce germination. Non-adherent cells were removed
by sev-eral washes with water. Adherent germ tube forms were
stainedwith 0.1% crystal violet for 5 min and washed three times
with dis-tilled water. Next, the 150 �L of isopropanol-0.02 N HCl
and 50 �Lof 0.25% SDS were added to each well to dissolve the
crystal vio-let. The absorbance of each well was measured using a
microplatereader at A590nm (ASYS UVM 340 Biogenet). Assays were
carried outtwice in three replicates.
In the case of biofilm formation, the assay was performed
asdescribed above, only the plates were initially incubated with
C.albicans and R. mucilaginosa cells for 12 h at 37 ◦C, washed and
incu-
y of gemini quaternary ammonium salts. Microbiol Res (2013),
bated with tested gemini-QAS. After incubation, non-adhered
cellswere removed by several washes. Wells were stained with
crystalviolet and the absorbance of each well was measured as
describedabove. The assay was performed twice in three
replicates.
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Table 1Minimal inhibitory concentration (MIC) of gemini
quaternary ammonium salts.
Compound Minimal inhibitory concentrations [�M] of gemini
surfactants for tested strains:
Saccharmyces cerevisiae Candida albicans Candida parapsilosis
Rhodotorula mucilaginosa
TMEG-6 Cl 800 800 320 160TMEG-8 Cl 160 80 40 10TMEG-10 Cl 10 40
10 10TMEG-12 Cl 320 800 500 20TMEG-12 Br 500 800 500 80TMEG-14 Cl
320 >1600 320 20
2
a6pIb
3
3
ewT((aatc
grCgb
3
tc8h(
TM
TMPG-10 Cl 20 80 TMPG-12 Cl 160 320 TMPG-12 Br 240 500
.12. Filamentous growth
To investigate whether gemini surfactants impact C. albicans
fil-mentation, the morphology of this strain was observed after 2,
4,
and 24 h incubations at 37 ◦C in YPG medium with a given
com-ound at final concentrations of MIC, 1/2 and 1/4 MIC. An
AXIO
mager A2 (ZEISS) microscope was used for observations. Scalear =
10 �m.
. Results
.1. Determination of minimal inhibitory concentration (MIC)
Among gemini quaternary ammonium salts with shorter spac-rs the
most active compound against the tested fungi was chloride,ith 10
carbon atoms within the alkyl chain (TMEG-10 Cl) (Table 1).
his compound exhibited antifungal activity at low
concentrations10–40 �M) against non-pathogenic (S. cerevisiae) and
pathogenicC. albicans, C. parapsilosis and R. mucilaginosa) fungi.
Shorteningnd elongation of the hydrocarbon chain caused the
decrease inntifungal activity. The comparison of bromide and
chloride withhe same alkyl chain length revealed that the compound
with thehlorine counterion has a stronger fungicidal effect.
The group of compounds with longer spacers included threeemini
surfactants, and the most active against fungi was chlo-ide with 10
carbon atoms within the hydrocarbon chain (TMPG-10l). Elongation of
the alkyl chain caused the decrease in antifun-al effect and
chlorides were more active against fungal cells thanromides
(TMPG-12 Cl, TMPG-12 Br) (Table 1).
.2. Hemolytic activity of gemini surfactants
Gemini surfactants with the lowest MIC values were tested
forheir hemolytic activity. It was shown that the compound with
8
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arbon atoms within the alkyl chain and the shorter spacer (TMEG-
Cl) did not exhibit hemolytic activity at MIC, or a three
timesigher concentration (Fig. 3). Elongation of the hydrophobic
chainto 10 carbon atoms) did not cause hemolysis at minimal
inhibitory
able 2utagenicity of gemini surfactants measured in the number
of revertants and mutagenic
Tested compound Concentration [�M] TA98
Colony n
PCa > 400 NCb 34 ± 7.8 TMEG-10 Cl 20 56 ± 8.5
40 33 ± 2.8 TMPG-10 Cl 20 24.5 ± 0.7
40 27 ± 2.8 a As positive control (PC) cisplatin (5 �g/plate)
was used.b As negative control (NC) bacterial dilution was added to
top agar without tested comc Mutagenic ratio (MR)–see Ames’ Test in
Section 2. The values are average from at lea
10 10240 40500 40
concentration, however with the increase of concentration
somedisintegration of erythrocyte membrane was observed.
Chloridewith the longer spacer (TMPG-10 Cl) caused the strongest
hemoly-sis. 10 �M of this compound slightly lysed erythrocytes, but
80 �M(MIC for C. albicans) effected in the high degree of hemolysis
(60%)(Fig. 3).
3.3. Cytotoxic activity of gemini surfactants
The cytotoxicity of gemini surfactants with the strongest
anti-fungal activity was tested with resazurin solution
(AlamarBlue).No cytotoxic effect against mitochondrial metabolism
manifestedwith the change of the well color from blue to pink. Our
resultsshowed that none of the studied compounds was cytotoxic at
theconcentrations equal and lower than MIC.
3.4. Mutagenic potential of gemini surfactants
The mutagenicity of the most active gemini-QAS–TMEG-10 Cland
TMPG-10 Cl, measured by Ames’ Test showed that none ofthe tested
compounds exhibit mutagenic potential. The ratio of therevertant
number to negative control (MR) was elevated in the caseof TMEG-10
Cl for the TA98 strain (1.6), however it never reached2.0 (Table
2).
3.5. Influence of cationic gemini surfactants on C.
albicanssensitivity to azoles and polyenes
C. albicans is a fungal pathogen causing skin and systemic
infec-tions. Azoles (e.g., fluconazole, itraconazole) and polyenes
(e.g.,amphotericin B) are the most commonly used in their
treatment.Pathogenic strains can acquire resistance to these
antibiotics bye.g., active efflux of the drug by the plasma
membrane transportersMFS and ABC.
y of gemini quaternary ammonium salts. Microbiol Res (2013),
Our study showed that gemini surfactants enhanced fungicidalthe
effect of azoles at lower concentrations than when the antibi-otics
were applied solely. Azoles caused a drop of about 40% in
C.albicans viability, and the addition of gemini surfactant
significantly
ratio (MR).
TA100
umber MRc Colony number MRc
> 11.0 > 400 > 2.0241.5 ± 2.1
1.6 254.5 ± 3.5 1.050.97 196 ± 16.9 0.81
0.72 192 ± 12.7 0.790.79 97 ± 2.8 0.4
pounds.st three tests.
dx.doi.org/10.1016/j.micres.2013.06.001
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ARTICLE IN PRESSG ModelMICRES-25569; No. of Pages 9E. Obłąk et
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-10 C
eaapsC
Fa
Fig. 3. Hemolytic activity of gemini-QASs: (a) TMEG
nhanced fungicidal effect. The compound that decreased C.lbicans
viability on the highest level was TMEG-8 Cl, which causedround a
2-fold increase of C. albicans sensitivity (Fig. 4c). The com-
Please cite this article in press as: Obłąk E, et al.
Antifungal
activithttp://dx.doi.org/10.1016/j.micres.2013.06.001
ound with 10 carbon atoms within its alkyl chains (TMEG-10
Cl)howed a much weaker effect. It exhibited only 20% inhibition of.
albicans growth in the presence of itraconazole, and 40% in the
ig. 4. The influence of cationic gemini surfactants (TMEG-10 Cl,
TMPG-10 Cl, TMEG-8 Cl) ond (d, e, f) polyenes (AMB–amphotericin B)
(mean ± sd, n = 3).
l; (b) TMPG-10 Cl; (c) TMEG-8 Cl (mean ± sd, n = 3).
case of fluconazole (Fig. 4a). The elongation of the spacer
(TMPG-10 Cl) caused the increase of C. albicans sensitivity to
azoles a 50%inhibition of C. albicans growth (Fig. 4b).
y of gemini quaternary ammonium salts. Microbiol Res (2013),
Amphotericin B, another tested antibiotic, belongs to thepolyene
class. It was shown that gemini surfactants with 10 car-bon atoms
within the alkyl chain, independently of spacer length,
n Candida albicans sensitivity to: (a, b, c) azoles
(ITR–itraconazole; FLU–fluconazole)
dx.doi.org/10.1016/j.micres.2013.06.001
-
ARTICLE IN PRESSG ModelMICRES-25569; No. of Pages 96 E. Obłąk
et al. / Microbiological Research xxx (2013) xxx– xxx
F TMEGn
stas
3m
namgmt
(5faC(
3
lttCMriags
ig. 5. Adhesion inhibition of: (a) C. albicans by TMEG-10 Cl;
(b) R. mucilaginosa by = 3).
trongly increased C. albicans sensitivity (about 10% of growth)
tohis antibiotic (Fig. 4d and e). The weakest fungicidal effect
withmphotericin B was observed for TMEG-8 Cl, since it caused
onlylight drop of C. albicans viability (Fig. 4f).
.6. Effect of gemini surfactants on C. albicans and
R.ucilaginosa adhesion
Due to their structure and properties gemini quaternary ammo-ium
salts coat solid surfaces (e.g., plastic or silicone). The
longerlkyl chains of the surfactants cause the compound to have
aore hydrophobic nature. As a result of hydrophobic
interactions,
emini-QAS might coat the surface and block the adhesion
oficroorganisms, which is the first stage of biofilm formation
and
he cause of infections, which are strongly resistant to
treatment.Our results showed that TMPG-10 Cl at high
concentrations
240 �M for C. albicans and 160 �M for R. mucilaginosa) caused
a0% reduction in cell adhesion (Fig. 5c and d). 160 �M of gemini
sur-actant TMEG-10 Cl inhibited the R. mucilaginosa adhesion to
50%nd C. albicans adhesion was 25% decreased by 240 �M of TMEG-10l
(Fig. 5a and b). TMEG-8 Cl did not inhibit the adhesion of
strainsdata not shown).
.7. Effect of gemini surfactants on C. albicans filamentous
growth
The ability of C. albicans to form filaments is one of the
viru-ence determinants, thus the influence of gemini surfactants
onhis process was investigated. Microscopic observations showedhat
the tested compounds: TMEG-8 Cl, TMEG-10 Cl and TMPG-10l inhibited
C. albicans filamentation at concentrations equal to 1/4IC (Fig.
6). The reduction of filament formation was already occur-
ing after 2 h incubation with the compounds, however
complete
Please cite this article in press as: Obłąk E, et al.
Antifungal
activithttp://dx.doi.org/10.1016/j.micres.2013.06.001
nhibition of filamentation was noted only after 6 h incubation.
Fil-mentation inhibition was observed also at 1/2 MIC and MIC
ofemini surfactants with the repression of bud formation (data
nothown).
-10 Cl, (c) C. albicans by TMPG-10 Cl, (d) R. mucilaginosa by
TMPG-10 Cl (mean ± sd,
Filamentous growth is one of the factors facilitating adhesionto
host tissues and abiotic surfaces. The blockage of C.
albicansfilamentation by gemini-QAS might be one of the mechanisms
con-ferring their antiadhesive activity.
3.8. Effect of gemini surfactants on C. albicans and
R.mucilaginosa biofilm removal
Some fungal pathogens are able to form biofilm, composed ofcells
with altered metabolism and surrounded by an extracellularmatrix,
which makes the whole structure resistant to antifungals.The data
indicates that monomeric quaternary ammonium salts areefficient in
biofilm eradication, thus we decided to study the impactof
gemini-QAS on fungal biofilm.
Our results showed (Fig. 7) that TMPG-10 Cl destroyed about60%
of biofilm formed by C. albicans and R. mucilaginosa at MIC (80and
10 �M respectively) (Fig. 7). A 5-fold increase in the
concentra-tion of this compound caused an 80% eradication of R.
mucilaginosabiofilm, and the concentration of 240 �M removed the
biofilm com-pletely. In the case of C. albicans biofilm, the
increase in compoundconcentration did not cause any further
eradication of the biofilm(Fig. 7c and d).
Shortening of the spacer length in the compound with 10 car-bon
atoms within the alkyl chain (TMEG-10 Cl) slightly
decreasedantibiofilm activity. It was shown that the MIC of this
compounddestroyed C. albicans and R. mucilaginosa biofilm by 50%
and 40%,respectively. The complete eradication of R. mucilaginosa
biofilmwas observed at concentration of 100 �M (Fig. 7a and b).
Theantibiofilm activity was not observed for TMEG-8Cl (data
notshown).
4. Discussion
y of gemini quaternary ammonium salts. Microbiol Res (2013),
Gemini surfactants are a new class of amphiphilic compoundsbuilt
from two classic surfactant moieties bound together by a spe-cial
spacer group (Yoshimura et al., 2012; Buse et al., 2011; Zhou
andZhao, 2009; Tehrani-Bagha et al., 2012). These compounds
appear
dx.doi.org/10.1016/j.micres.2013.06.001
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ARTICLE IN PRESSG ModelMICRES-25569; No. of Pages 9E. Obłąk et
al. / Microbiological Research xxx (2013) xxx– xxx 7
F 4 MICb
timgve
re
attwpGQsp
ig. 6. Impact of gemini surfactants (TMEG-8 Cl, TMEG-10 Cl,
TMPG-10 Cl) at 1/ar = 10 �m.
o be excellent for creating complexes with DNA and to be
effectiven mediating transfection. Due to their construction, DNA
carrier
olecules built from gemini surfactants are able to deliver to
cellenes of almost any DNA molecule size, which is impossible
foriral gene delivery systems (Mc Gregor et al., 2001; Pullmannovat
al., 2012).
Gemini quaternary ammonium salts exhibit stronger antibacte-ial
and antifungal activity in comparison to mono-QAS (Lachowiczt al.,
1992, 1995; Shirai et al., 2012; Tisher et al., 2012).
A series of gemini quaternary ammonium salts with differentlkyl
chain lengths (6–14 carbon atoms), spacer lengths and coun-erions
(bromide or chloride) was synthesized. The data indicatehat
monomeric quaternary ammonium salts with 12 carbon atomsithin the
alkyl chain are the most active against yeast and gram-ositive
bacteria (Obłąk and Krasowska, 2010; Obłąk et al., 2002;
Please cite this article in press as: Obłąk E, et al.
Antifungal
activithttp://dx.doi.org/10.1016/j.micres.2013.06.001
ilbert and Moore, 2005). Our results showed that among gemini-AS
chlorides with 10-carbon hydrophobic chains and shorter
pacers (TMEG-10 Cl) had the strongest activity against yeast
andathogenic fungi. Elongation of the spacer (TMPG-10 Cl)
slightly
Fig. 7. The influence of TMEG-10 Cl on biofilms: (a) C.
albicans; (b) R. mucilaginosa an
on filamentous growth of C. albicans after 6 and 24 h incubation
at 37 ◦C. Scale
decreased the antifungal effect. Due to their strong activity
againstfungi, these compounds could be applied as potential
fungicides.Our further studies concerned the compounds with the
lowest MICvalues (TMEG-10 Cl, TMEG-8 Cl and TMPG-10 Cl). It was
shown thatcationic gemini surfactants with shorter spacers
exhibited weakhemolytic activity (about 15%). The compound with 8
carbons inthe alkyl chain did not cause significant hemolysis even
at concen-tration three times higher than MIC. Similar results were
obtainedfor pirydynium-based gemini quaternary ammonium salts
(Shiraiet al., 2009), where the concentration causing 50% hemolysis
wasmuch higher for the surfactant with 8 carbons within the
hydrocar-bon chain in comparison with the remaining compounds.
Amongtested gemini surfactants, the compound with 10 carbon
atomswithin the alkyl chain did not show any significant hemolytic
activ-ity at MIC, however a 2-fold increase in concentration caused
a
y of gemini quaternary ammonium salts. Microbiol Res (2013),
high degree of erythrocyte membrane disruption. The elongationof
the spacer (TMPG-10 Cl) also increased hemolytic activity (50%of
hemolysis in MIC), which excludes the potential application ofthis
compound as a drug in internal mycosis treatment. Potential
d TMPG-10 Cl on biofilms: (c) C. albicans; (d) R. mucilaginosa
(mean ± sd, n = 3).
dx.doi.org/10.1016/j.micres.2013.06.001
-
ING ModelM8 ical Re
frtAa
aopmflmdA2a2stAtmiOscQm2cs1
nd2pofmtlptC
omchibwaaeobem
tc2as
ARTICLEICRES-25569; No. of Pages 9E. Obłąk et al. /
Microbiolog
ungicides should not exhibit cytotoxic effects. Our
preliminaryesults showed that gemini quaternary ammonium salts were
notoxic for the mitochondrial metabolism of S. cerevisiae.
Moreover,mes’ test showed that studied surfactants had no
mutagenicctivity.
Common usage of antifungals raises multidrug resistancemong
microorganisms. Pathogenic fungi have developed numer-us mechanisms
of resistance, which allow them to survive in theresence of toxic
agents (White et al., 1998). The drugs most com-only applied in
mycosis treatment are azoles (e.g., ketoconazole,
uconazole), which inhibit the biosynthesis of ergosterol, one of
theain lipids building the plasma membrane. The resistance to
these
rugs occurs via different mechanisms, e.g., the overexpression
ofBC and MFS pumps that actively efflux antibiotics (Sanglard et
al.,009). Currently substances, which would enhance the
antifungalctivity of drugs (e.g., azoles) are being investigated
(Nyilasi et al.,010; Ahmad et al., 2010; Kiraz et al., 2010). The
results of our studyhowed that gemini-QAS strongly increased C.
albicans (in plank-onic form) sensitivity to azoles (fluconazole
and intraconazole).nother popular class of antibiotic are polyenes
(nystatin, ampho-
ericin B), which (as amphiphilic drugs) intercalate into the
plasmaembrane, creating channels for cellular components (mainly
K+
ons) and disrupting the proton gradient (te Welscher et al.,
2012).ur studies regarding the influence of gemini-QAS on C.
albicans
ensitivity to amphotericin B showed that the combination of
bothompounds significantly decreased C. albicans viability.
Gemini-AS, similarly to polyenes, affect the plasma membrane,
causingono- and divalent cation, as well as ATP, leakage (Shirai et
al.,
009; Palermo et al., 2011). The combined activity of these
twolasses of substances might strongly disrupt plasma
membranetructure, decreasing the survival of fungal cells (Ramos et
al.,996).
C. albicans is one of the most common human pathogens causingot
only superficial mycosis but also systemic infections,
especiallyangerous for immunocompromised patients (Pfaller and
Diekema,007). C. albicans may grow in different forms: yeast,
pseudohy-hal and hyphal. The switch between different types of
growth isne of the virulence determinants, because it can lead to
biofilmormation inside the host organism. The first stage in
biofilm for-
ation is the adhesion of fungal cells to the surface (e.g.,
intestinalissue). During biofilm maturation the cells produce an
extracellu-ar matrix, composed of carbohydrates, proteins and
phosphates,rotecting biofilm from phagocytes and preventing drug
penetra-ion into the biofilm structure (Blankenship and Mitchell,
2006;uéllar-Cruz et al., 2012).
Biofilm formation is also common for Rhodotorula spp., thatften
colonize medical devices such as catheters or hemodialysisachines
(Zaas et al., 2003). Gemini surfactants, as amphiphilic
ompounds, might coat the biotic and abiotic surfaces (due
toydrophobic interactions) and block the adhesion of
microorgan-
sms similarly to the action of some biosurfactants isolated
fromacteria (Janek et al., 2012). Our results showed that
gemini-QASith 10 carbon atoms within the alkyl chain (with both
shorter
nd longer spacers) inhibit the adhesion of R. mucilaginosa and
C.lbicans to the polystyrene surface at high concentrations.
Short-ning of the alkyl chain to 8 carbon atoms caused the
decreasef antiadhesive activity, probably due to hydrophobic
interactionseing too weak. The application of gemini-QAS as
adhesion block-rs would reduce the risk of biofilm arising on the
host tissues andedical devices.The capacity for filamentous growth
(hypha formation) is one of
he virulence factors in C. albicans. Filamentation promotes
fungal
Please cite this article in press as: Obłąk E, et al.
Antifungal
activithttp://dx.doi.org/10.1016/j.micres.2013.06.001
ell adhesion and biofilm maturation (Whiteway and
Bachewich,007). The inhibition of hypha production lowers the risk
of C.lbicans infections (Messier and Grenier, 2011). To date
numerousubstances, which block the switch between yeast and
filamentous
PRESSsearch xxx (2013) xxx– xxx
forms were identified, examples being whey-derived fatty
acids(linoleic, arachidonic) and capric acid isolated from
Saccharomycesboulardii (Clement et al., 2007; Murzyn et al., 2010).
Gemini qua-ternary ammonium salts are also effective inhibitors of
C. albicansfilamentous growth. Our results showed that chlorides
with 10 and8 carbons in the hydrocarbon chain almost completely
inhibitedfilament formation at concentrations 4- fold lower than
MIC. Theusage of these compounds against C. albicans cells would
signifi-cantly reduce their virulence, and biofilm creation.
The biofilm is extremely stable and resistant to
numerousantimicrobial drugs (Ramage et al., 2005). Infections of
medicaldevices (catheters, implants) by biofilm-forming
microorganismsmean that the infected device needs to be removed and
replaced.Monomeric quaternary ammonium salts are highly effective
inbacterial biofilm eradication. It was shown that
dimethylbenzy-lammonium chloride was strongly active against
biofilm formed byStaphylococcus epidermidis. This compound
penetrated inside thebiofilm structure, but also changed the
properties of the extracel-lular matrix, making it weaker and fluid
(Davison et al., 2010).
Our results indicated that gemini quaternary ammonium
saltsexhibit strong activity against fungal biofilm. Chlorides with
10carbon atoms within the alkyl chain (TMEG-10 Cl and TMPG-10 Cl)
were highly reactive, especially against biofilm formed byR.
mucilaginosa, because their minimal inhibitory
concentrationseradicated about 50% of the generated biofilm.
Due to rising multidrug resistance among microorganisms,there is
a strong need to search for new substances that wouldinhibit their
growth and reduce the virulence. Currently studiedgemini quaternary
ammonium salts, due to their unique properties,could be good
candidates for application as fungicides or disinfec-tants in order
to overcome multidrug infections, often caused bybiofilm
formation.
Acknowledgements
The work was supported by the Polish Ministry of Science
andHigher Education grant No N N209 337 737 and by the
statutoryresearch of the Institute of Genetics and Microbiology at
Universityof Wrocław. Authors’ contribution: the study of
biological activ-ity of gemini-QASs–E.O. and A.P.; synthesis of
gemini-QASs–J.Ł.;equipment availability–A.K. We are grateful to
Prof. StanisławWitek (Wrocław University of Technology) for helpful
comments.
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