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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

Please cite this article in press as: Obłąk E, et al. Antifungal activithttp://dx.doi.org/10.1016/j.micres.2013.06.001

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

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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


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


Candida albicans ATCC 90028, Candida parapsilosis IHEM 3270,Rhodotorula mucilaginosa IHEM 18459 and Saccharomyces cerevisiae�1278b.

dx.doi.org/10.1016/j.micres.2013.06.001

ARTICLE IN PRESSG ModelMICRES-25569; No. of Pages 9E. Obłąk et al. / Microbiological Research xxx (2013) xxx– xxx 3

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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


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-


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.


ARTICLE IN PRESSG ModelMICRES-25569; No. of Pages 94 E. Obłąk et al. / Microbiological Research xxx (2013) xxx– xxx

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

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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


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.


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.



-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-


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).


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)


ARTICLE IN PRESSG ModelMICRES-25569; No. of Pages 96 E. Obłąk et al. / Microbiological Research xxx (2013) xxx– xxx

F TMEGn

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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


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


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



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;


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


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).


ING ModelM8 ical Re

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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


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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