Infectious Diseases in Obstetrics and Gynecology 8:124-137 (2000) (C) 2000 Wiley-Liss, Inc. Antifungal Activity of Local Anesthetics Against Candida Species Cidfilia Pina-Vaz,,2,3. Acficio Gonalves Rodrigues,1,2 Filipe Sansonetty,2 J. Martinez-De-Oliveira,4 Ant6nio F. Fonseca,1 and Per-Anders Mfirdh5 1Department ofMicrobiology, Porto SchoolofMedicine, University ofPorto, Porto, Portugal elnstitute ofPathology andMolecularImmunology ofPorto University, Porto, Portugal 3Institute ofMolecularandCellBiology, Porto SchoolofMedicine, University ofPorto, Porto, Portugal 4DepartmentofGynaecology, Porto SchoolofMedicine, University ofPorto, Porto, Portugal SDepartmentofObstetrics andGynaecology, Lurid University, Lurid, &seden ABSTRACT Objective: To evaluate the activity ofbenzydamine, lidocaine, and bupivacaine, three drugs with local anesthetic activity, against Candida albicans and non-albicans strains and to clarify their mechanism ofactivity. Methods: The minimal inhibitory concentration (MIC) was determined for 20 Candida strains (18 clinical isolates and two American Type Culture Collection strains). The fungistatic activity was studied withthefluorescentprobe FUN-1 andobservationunderepifluorescence microscopy and flow cytometry. The fungicidal activity of the three drugs was assayed by viability counts. Membrane alterationsinducedintheyeastcellswere evaluatedby stainingwithpropidiumiodide, byquantitation ofintracellularK leakage andbytransmissionelectronmicroscopyofintactyeast cells and prepared spheroplasts. Results: The MIC ranged from 12.5-50.0 pg/mL, 5.0-40.0 mg/mL, and 2.5-10.0 mg/mL for benzydamine, lidocaine, and bupivacaine, respectively. The inhibitory activity ofthese concentra- tions could be detected with thefluorescentprobe FUN-1 afterincubationfor60 minutes. Avery fast fungicidal activity was shown by 0.2, 50, and 30 mg/mL ofbenzydamine, lidocaine, and bu- pivacaine, respectively. Conclusions: At lower concentrations, the tested drugs have a fungistatic activity, due to yeast metabolic impairment, while athigher concentrations they are fungicidal, due to direct damage to the cytoplasmic membrane. Infect. Dis. Obstet. Gynecol. 8:124-137, 2000. (C)2000Wiley-Liss,Inc. KEYWORDS lidocaine, bupivacaine, benzydamine, Candida, fungicidal andidaalbicansis acommon causative agentof growing number ofimmunocompromised patients, mucosal fungal infections,1,z which are diffi- e.g., due to iatrogenic measures and to persons in- culttotreatand tend torecur.1,3There has beenan fected by human immunodeficiencyvirus. The in- increasing rate of candidosis, particularly due to a creasing use of antifungal drugs, both for prophy- Grantsponsor:ComisstodeFomentodaInvestigagoemCuidadosde Safide-Minist6riodaSatide;grantnumber: 113/96. Grant sponsor: Praxis XXI: [Fundagfio para a Ciencia e Tecnologia, Lisboa, Portugal]; grant number: PSAU/C/SAU/ 0014/96. *Correspondence to: Cidfilia Pina-Vaz, Department of Microbiology, Porto School of Medicine, 4200 Porto, Portugal. E-mail: [email protected] Received 22 November 1999 Clinical Study Accepted 25 February 2000 LOCAL ANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. TABLE I. MICs and LCs0s for benzydamine, lidocaine, bupivacaine, and fluconazole of Candidastrains MIC Benz. LCso Benz. MIC Lid. LCso Lid. MIC Bup. LCso Bup. MIC Flu. Strains Isolates (lg/mL) (lg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (lg/mL) C. albicans M36 Vaginal 12.50 5.0 2.50 >64 C. albicans H33 Vaginal 6.25 5.0 2.50 C. albicans SCO Vaginal 6.25 2.5 1.25 C. albicans M9 Vaginal 6.25 2.5 1.25 C. albicans Ser5 Vaginal 12.50 5.0 2.50 16 C. albicans H65 Vaginal 12.50 2.5 1.25 >64 C. albicans M28 Vaginal 25.00 2.5 1.25 2 C. albicans H38 Vaginal 12.50 5.0 2.50 C. albicans 10231 ATCC 12.50 36.38 5.0 19.96 2.50 20.06 C. albicans H37 Bronchial wash 12.50 49.14 10.0 32. 5.00 3.621 >64 C.glabrata H30 Vaginal 25.00 54.59 40.0 354.07 10.00 263.83 >64 C.glabrata H16 Vaginal 50.00 87.59 10.0 49.45 5.00 39.56 64 C. krusei H9 Blood 25.00 58.70 10.0 22.14 2.50 15.12 >64 C. krusei H32 Blood 25.00 54.38 10.0 20.73 2.50 12.72 >64 C. tropicalis 13803 ATCC 12.50 48.21 10.0 61.23 2.50 25.30 4 C. tropicalis H18 Blood 25.00 67.73 5.0 46.63 2.50 16.59 4 C.guilliermondiMAT24 Vaginal 12.50 40.31 10.0 51.62 2.50 28.61 4 C.guilliermondi MAT23 Vaginal 25.00 58.29 10.0 54.40 5.00 31.68 2 C. lusitaniae H22 Vaginal 25.00 74.42 10.0 54.40 5.00 102.15 C. lusitaniae H54 Bronchial wash 50.00 88.23 5.0 27.93 2.50 16.88 aBenzydamine, Benz.; lidocaine, Lid.; bupivacaine, Bup.;fluconazole, Flu. lactic and therapeutic purposes, has led to growth phase cells) or subcultured in Sabouraud the emergence of resistant strains.4,s This situa- broth tothe middleofthe exponentialgrowthphase. tion calls for the search for alternative antifungal Antifungal Drugs drugs. Many nonantibiotic drugs including antidi- Benzydamine was obtained from Lepori Angelini uretic, antidiabetic, [3-blockers, psychotherapeutic, (Rome, Italy). Lidocaine and bupivacaine were and nonsteroidal anti-inflammatory molecules pos- purchased from Sigma (St. Louis, MO). sess an antimicrobial action, which has generally been regarded as a side effect6 and therefore ne- Incubation of Yeast Cells With Drugs glected for potential clinical use. Benzydamine, li- Yeastcells in stationaryphasewere resuspended in docaine, and bupivacaine are known nonantibiotic 10 mmol/L sodium N-2-hydroxyethylpiperazine- antimicrobials. Topical use of these anesthetic N-2-ethanesulfonic buffer (HEPES, pH 7.2), drugs may be useful in the management of cuta- supplemented with 2% glucose (GH solution), at a neous and vaginal candidosis. We studied the ac- densityof x 106-5 x 106cells/mL,with orwithout tivity and mechanism of action of benzydamine, serial concentrations of the drugs (see legends to lidocaine, and bupivacaine against C. albicans and figures). Incubations were carried outat35C, with non-albicans strains. shaking at 200 strokes/min. At the end of the in- cubation, the cells were centrifuged for 10 minutes MATERIALS AND METHODS at 1800g, and the antifungal activity of the drugs Candida Strains was assayed by viability counts and by staining Twenty Candida strains were used: 18 clinical iso- with the fluorescent probes Propidium Iodide (PI) lates and two American Type Culture Collection and FUN-I, as described below. (ATCC) strains (Table 1). The yeasts were kept at Determination of Minimal -70C in Brain-Heart broth (Difco Laboratories, InhibitoryConcentrations Detroit, MI) with 5% glycerol until tested. For each experiment, the strains were subcultured The minimal inhibitory concentrations (MICs) of twice on Sabouraud agar (Difco) for 24 hours at the antifungals were determined by a macrodilu- 35C and either resuspended in saline (stationary tion test, according to the reference method INFECTIOUSDISEASESINOBSTETRICSANDGYNECOLOGY 125 LOCALANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. (M27-A protocol) of the National Committee for the exponential growth phase. The yeasts were Clinical Laboratory Standards.7 washed twice with saline and exposed at 35C for 10 minutes to the assayed drugs dissolved in saline Yeast Cell Counts atindicatedconcentrations. After5 and 10minutes, The total number ofyeasts in the suspensions was treated and control suspensions were filtered determined in a Neubauer hemocytometer (Agar through 0.45 pm Millipore filters (Millipore, MA). Scientific Ltd, Stansted, UK). Enumeration of vi- The filtrates were assayed for K using a K+- able yeast cells in the untreated control suspen- sensitive glass electrode connected to a Spotlyte sions and in those exposed to local anesthetics or analyzer (Menarini Diagnostics). The values are sodium azide was carried out by counting colony- presented as the percentage of K leaked in com- forming units (CFU) after plating serial dilutions parison to that from cells boiled for 30 minutes.9’1 (in saline) of the suspensions on Sabouraud agar Viabilitycounts and the percentage ofcells stained plates. The number ofcolonies was counted after by PI were also determined in the suspensions 48 hours ofincubation at 35C. used for the K leakage assays. The leakage ofK was also analyzed for Candida cells treated with 20 Studies of Membrane Damaging mM sodium azide, orwith 2pg/mL ofamphotericin Two independent procedures were used to assess B, for 10 minutes. thecapacityofbenzydamine, lidocaine,and bupiv- Assays of MetabolicVitality acaine to damage the fungal cytoplasmic mem- brane. One relied on the use of the membrane- The processingofthe fluorescentprobe FUN-1 by impermeable fluorescent dye PI. Previous the yeast cells was used to detect nonlethal meta- experiments have been carried out to optimize the bolic alterations. Two methods to assess FUN-1 flow cytometric conditions that were used in the A current study (Pina-Vaz et al, in press). That is, SINGLE PBETER optimal resultswere obtainedwhenusing 106yeast cells/mL, stained with lpg/mL of PI, for 30 min- utes in 0.05 mol/L sodium HEPES buffer, pH 7.2, at room temperature in the dark. Incubation with lpg/mL of PI under the above-mentioned condi- tions had no toxicity to Candida cells (as deter- mined by viability counts) and stained 100% of Candida cells killed by boiling for 30 minutes F12Log (Pina-Vaz et al., in press). For each sample, the percentage ofPI-positive cells was determined by flow cytometry as described in detail below. From B these values, the concentration of the assayed SINGLE PRJETER drugs resulting in 50% PI-positive cells was calcu- lated accordingtoa linearregression equation. The PI staining was found to be an adequate indicator ,,,,,,, of cell death (see "Results"). Therefore, we con- sidered those drug concentrations causing half of the cells to be stained as representing median le- thal concentration (LC50). ,..,;;,i, 18 188 1888 The second method we used to study cytoplas- F12Log mic membrane damage estimated the leakage of intracellular K from the yeast cells. Because the Fig. I. Single parameter histograms of FUN-I stained C. intracellular accumulation ofK is higher in yeasts albicans,ATCCstrain 10231 cellsafter hourofincubation with benzydamine (A) or bupivacaine (B). a: autofluores- in the exponential growth phase,8 Candida cells cence (without FUN-I); b: untreated control; :treatment grown at 35C in Sabouraud broth supplemented with 12.5 tsg/mL of benzydamine; d: treatment with 2.5 with0.5% KzHPO wereharvested atthemiddleof mg/mL ofbupivacaine. 4 126 INFECTIOUSDISEASESINOBSTETRICSANDGYNECOLOGY LOCAL ANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. 100 6o Benz. 100g/ml Benz. 50xtg/rnl o Benz.25j.tgtrnl 2o 6 1’ 2’0 ’5 ’0 o Ttme rntnurea) 100- 80 60 Benz. 100tg/rnl Benz. 50tg/rnl o Benz. 25jttg/ml 20 Time rninutes) 100- . 0 LID 10mg/rnl 60 LID20mg/rn] 4o LID 30rng/ml 2o o ; , ’o ’ o ,o o Time rninurea,) Fig. 2. Percentage ofunviable (A, C, E) and Pl-positive, (B, D, F) C. albicans, ATCC strain 10231 cells exposedfor 30 rain to increasing concentrations ofbenzydamine (benz.) (A, B), lidocaine (LID) (C, D) or bupivacaine (BUP) (E, F). processing were used. Biochemically active cells, lution were incubated with 0.5 pM ofFUN-1 (Mo- stained with this fluorescentmembrane-permeable lecular Probes Europe BV, Leiden, Netherlands) dye, exhibit under fluorescence microscopy or- for 30 minutes at 30C in the dark. To evaluate ange/red cylindrical intravacuolar structures CIVS formation, the FUN-1 stained cells were (CIVS), while nonviable cells or viable cells with mounted on microscope glass slides with the anti- severely impaired metabolism do not show these fading Vectashield Mounting Medium (Vector structures.11 Metabolically impaired yeast cells Laboratories, Burlingane, CA). The percentage of showan increased intracellularaccumulation ofthe yeastswith CIVSwas determined byobserving200 probe, which can be detected by flow cytometry,lz cells under epifluorescence microscopy in a Leitz Untreated and treated yeastsuspensions in GH so- Laborlux K (Leica, Buffalo, NY) microscope fitted INFECTIOUSDISEASESINOBSTETRICSANDGYNECOLOGY 127 LOCALANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. I00 8o LID 10rnglml LIDZOmg/ml LID 5Orngfml 0 o Tme mnutes) 100 . 60 BUP 10rng/rnl BUP20rnglml 40 BUP30mglml 20 Time rninutes) I00- @e 8o BUP Orng,."ml BUP20rng/rnl = BUP30rng/ml 20 0 5 0 15 20 25 30 Tme rnnues) Fig. 2. Continued. with a mercury 50-W lamp, a BP 450-490-nm ex- without the fluorochrome (autofluorescence, as a citation filter and a LP 515-nm emission filter. To control). quantify the intracellular concentration of FUN-I, Spheroplast Formation flow cytometry was used as described below. Spheroplasts of C. albicans, ATCC strain 10231, Flow Cytometry were obtained by enzymatic digestion of the cell The suspensions were analyzed at 620 nm (FL3) wallwith Lyticase(BoehringerMannheim,Cat. No for PI and at 575 nm (FL2) for FUN-l, in a Beck- 1372464, Mannheim, Germany).13 Incubation was man Coulter XL-MCL (Hialeah, FL) flow cytom- made at 35C in YEPD medium (1% yeast extract, eter, equipped with a 15-mV argon laser with and 2% bacto peptone, and 2% glucose), containing 128 INFECTIOUSDISEASES1NOBSTETRICSANDGYNECOLOGY LOCALANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. A C.elbieensATCC C.elbicans H;57 100 C.kruzei C.krusei H9 C.glabrata H 6 60 C.cjlabrete HSO 40 -" C.lusitanee H22 C.Iusitanee H54 20 C.tropicelis H 8 0 ATCCtropicalis 0 50 100 150 200 C.gui11 I’-I6T25 Benzidamine(Ixg/ml) C.gui11 NAT24 B C.albicans6TCC 100 C.albicons H37 C,kruaei H52 80 C.krusei H9 C.glabrata H16 C.glabrata H:50 C,1usitenea H22 C,lusianea H54 20 C.tropicelis HI8 ATCCtropicalis 0 0 20 0 40 SO C.guill MAT25 Lidocaine(mg/ml) C.guill PLAT24 C C,elbicansATCC C.elbicans H37 100 = C.kruaei H32 BO C.krusei H9 C.glabrata H 6 60 C.glabrata H30 40 C.1usitanee H22 C.1usitanee H54 20 = C.tropicalis HI8 0 ATCCtropicalis 0 10 20 = C.guill MAT2:3 Bupivacaine(mg/ml) x C.guil] M,T24 Fig. 3. Percentage of Pl-positive cells in suspensions ofCandida strains exposed for 60 min to different concentrations of benzydamine (A), lidocaine (B), or bupivacaine (C). yeast cells grown to middle of exponential phase concentration of10 units/107 cells, and the suspen- (about x 107 cells/mL). The cells were collected sion was incubated at 30C with gentle, occasional by centrifugation at 1800gfor 10 min, washed once shaking. Spheroplast production was monitored by with water and oncewith 1.4 mol/L sorbitol before phase contrast microscopy, assessing the lysis of the pellets were resuspended at a concentration of yeast cells exposed to 5% sodium dodecyl sulfate x 107-5 x 107 cells/mL in 0.04 M HEPES buffer (SDS). Yeasts unexposed to Lyticase did not lyse (pH 7.4), 0.5 M MgC1z, and 0.5% mercaptoethanol with SDS. When most Candida cells were con- (Sigma) in 1.4 M sorbitol. Lyticase was added at a verted into spheroplasts, the suspension was can- INFECTIOUSDISEASESINOBSTETRICSANDGYNECOLOGY 129 LOCALANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. A 100- 0 BUP Omg/rnl LID 50mglml 60 Benz 500j.tg/ml 40 NaazideZOmM 0 0 5 10 Time(m|nutes) B 100- 80 BUP :50rng/rnl LID 50mg/ml : Benz OOj.tg/rnl 40 Naazid 20ram 20 0 5 10 Timo(minutos) C 100- BUP :50rngtml 80 LID E;Omglml x 60 Benz :500j.tgfml Noazide ZOmM control 20 0 Time (minutes) Fig. 4. Exponential phase C. albicans,ATCC strain 10231 cells exposedfor I0 min to 0.3 mg/mLofbenzydamine (Benz), 50 mg/mLoflidocaine (LID), 30 mg/mLofbupivacaine (BUP) or20 mM sodium azide (Naazide). Inthesamesuspensions,three parameters were determined, i.e., the percentage of nonviable cells (determined by CFU counts) (A), the percentage of PI-positive cells (B), and K efflux (as percent ofthe total intracellular K/) (C). trifuged at 1800gfor 10 minutes and the pellet re- spheroplasts were studied. Blastoconidia of un- suspended in GH medium supplemented with 1.4 treated and treated cells ofC. albicansstrain ATCC M sorbitol with indicated concentrations ofthe as- 10231, were prefixed with 2.5% glutaraldehyde in sayed antifungal drugs. 0.1 M cacodylate buffer, pH 7.2, followed bywash- ing in the same buffer. The cells were then fixed Transmission Electron Microscopy with 1.5 % potassium permanganate in water for To analyze the ultrastructural alterations induced hour,14 followed by washing with water and post- by the tested drugs, C. albicans blastoconidia and fixation with aqueous 1% uranyl acetate for 30 130 INFECTIOUSDISEASESINOBSTETRICSANDGYNECOLOGY LOCAL ANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. minutes.1S Samples ofcontrol and treated sphero- Treatment of C. albicans, ATCC strain 10231, plasts were prefixed with 2.5% glutaraldehyde (1 with increasing concentrations of the anesthetics volume of 25% glutaraldehyde stock solution resulted in a dose-dependent fungicidal effect added to 9 volumes of spheroplast suspensions). (Fig. 2A, C, and E). Staining with PI showed that After at least 4 hours at room temperature, the the numberofPI-positivecellscorrelatedwellwith sampleswerewashedwith0.1 Mcacodylatebuffer, the number ofnonviable cells (Fig. 2B, D and F). pH 7.2, and fixed overnight at room temperature Figure 3 (A, B, and C) shows the effect of a 60- with 1% OsO4 in 0.1 M acetate-veronal buffer, pH minute exposure of some of the Candida strains 7.0, supplemented with 10 mM calcium chloride.16 investigated to increasing concentrations of the After washing with water, the cells were postfixed drugs. The drug concentrations resulting in LCso with aqueous 1% uranyl acetate for 30 minutes at were calculated for each strain (Table 1). The cor- room temperature,is Fixed intact cells and sphero- relation coefficient between the LCs0 ofeach drug plasts were dehydrated in ethanol and embedded for the 12 strains tested was r 0.073 when com- in Epon (TAAB Aldermaston, Berks, UK).7 Ultra- paring benzydamine with lidocaine, r 0.001 for thin sections were cut with an LKB Ultratome III benzydamine vs. bupivacaine, and r 0.955 for li- microtome (LKB-Produkter AB, Stockolm-Bromma, docaine vs. bupivacaine. Sweden) andcontrastedwith uranyl acetatefollowed The concentrations of the three drugs causing by lead citrate.16 To improve visualization of ribo- an extensive rate of killing (Fig. 4A) and perme- somes in the spheroplasts, sections were treated ability to PI (Fig. 4B) in C. albicans, ATCC 10231, with 3% hydrogen peroxide for 10 minutes before cells induced a quick and extensive leakage ofin- lead citrate staining.7 Observations and micro- tracellular K (Fig. 4C). A similar pattern of K graphs were done with a Zeiss EM 10C electron leakage was seen with the membrane-active anti- microscope (Carl Zeiss, Oberkochen, Germany). fungal amphotericin B, with 85.7% and 94.1% of intracellular K being lost after treatment ofC. al- Statistical Analysis bicans ATCC 10231 cells with 2 lag/mL of the an- Correlation coefficients (r) were calculated using tibiotic for 5 and 10 minutes, respectively. Treat- the Anova program (program Statistica for Win- ment ofC. albicans, ATCC strain 10231 cells, with dows, Stat Soft, CA). 20 mM sodium azide for 10 minutes resulted in RESULTS extensivecell death (Fig. 4A)butdid notmakethe The three drugs tested inhibited growth ofall Can- cells permeable to PI (Fig. 4B), nor did it induce a dida strains studied, with MICs ranging from 6.25 significant K leakage (Fig. 4C). Candida glabrata to 50.0 lag/mL for benzydamine, from 1.25 to 40.0 strain H30wasthe strain mostresistanttothe three mg/mL forlidocaine, and from 2.5 to 10 mg/mL for drugs assayed. When it was treated with high con- bupivacaine (Table 1). The correlation coefficients centrations of lidocaine, no K leakage was seen between the MIC of each drug for the 20 strains (not shown). studied were good when comparing lidocaine with Transmission electron microscopy showed that bupivacaine (r 0.905), but not for benzydamine exposureofC. albicans, ATCC strain 10231, tocidal vs. lidocaine (r 0.212) and not for benzydamine concentrations of the anesthetics induced severe vs. bupivacaine (r 0.328). Short exposure of C. alterations ofthe cytoplasmic organelles (Fig. 5). albicans strain ATCC 10231 to the MIC ofeach of Exposure ofblastoconidia ofC. albicans, ATCC the three drugs did not result in anycell death and strain 10231, to Lyticase--under the described did not make the cells permeable to PI or induce conditions--resulted in detergent-sensitive sphe- any significant K leakage (not shown). However, roplasts, which often had loose cell wall remnants under these conditions, the drugs significantly im- (Fig. 6A). Only occasionally completely wall-free paired the vitality of the Candida cells, as was protoplasts were found. Spheroplasts not exposed shown by FUN-1 experiments. Thus, yeast cells to the drugs were intact and showed numerous ri- exposed for 1 hourto the drugs atthe MIC did not bosomes and normal intracellular organelles, i.e., form CIVS (not shown), while an increase in the the nucleus and the mitochondria (Fig. 6, Aand B) intracellular fluorescence was detected by flow cy- and intactcell membraneswithacontinuous triple- tometry (Fig. 1). layered profile (Fig. 7A). On the contrary, when INFECTIOUSDISEASESINOBSTETRICSANDGYNECOLOGY 131 LOCALANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. .. Fig. 5. Transmission electron microscopy ofuntreated C. albicans, ATCC strain 10231, cells afterfixation with glutaral- dehyde/permanganate/uranyl. N, nucleus; M, mitochondria; R, endoplasmic reticulum; V, vacuole. Section contrasted with uranyl-lead (x 13,900). B. A Candida cell from the same sample as in Fig. 5A but treated for 60 min with 0.3 mg/ml_ of benzydamine. Notice the severe intracytoplasmic disorganization, including the absence of mitochondria and endoplasmic reticulum. Section contrasted with uranyl-lead. (x 13,900). treated for 10 minutes with fungicidal concentra- of hour, therebyallowingfora morerapid assayas tions of the drugs most sphcroplasts were lysed compared with the conventional protocol for MIC (Fig. 6, C, D, and E), showing severely damaged determination requiring 24 hours. cell membranes with gross fractures or extensive The followingresults pointto the mechanism of solubilization (Fig. 7, B and C). Ultrastructural im- the fungicidal action ofthe drugs being due to di- ages of plasma membrane splitting were seen in rect damage to the yeast cytoplasmic membrane. some drug-exposed yeast cells (Fig. 7C). Thus, Candida cells exposed to fungicidal concen- trations ofthe drugs quickly become permeable to DISCUSSION the membrane-impermeable fluorochrome PI. Localanestheticdrugspossessantibacterialand an- This probe has previously been used to evaluate tifungal properties.9,18-22 However, theirantifungal membrane permeabilityinyeasts and isconsidered mechanisms have not yet been elucidated. a good marker for cell death associated with mem- Using a combination of complementary meth- brane alterations,e,e4 Quantitation of K leakage odologies, we found the drugs to have a marked from microorganisms, both from bacteria9,es and antifungal activity on C. albicans that ranged from yeasts,l,e6-e9 has also been used to evaluate mem- growth inhibition to complete loss of viability. brane damage byvarious compounds. Yeastcells in Growth inhibition can bc detected byconventional the exponential growth phase are known to accu- protocols for MIC determination, as demonstrated mulate K intracellularly, with concentrations as in our study. We found that the fungistatic activity high as 220 mM.s This cation is quickly lost to the ofthe anesthetics could also be detected, with ad- extracellular milieu when the selective membrane vantages, by use ofFUN-1. Yeast cells exposed to permeabilityis lost.,e6-e9Avery quickand exten- MIC of the drugs showed both inhibition of con- sive K leakage was induced by fungicidal concen- version ofthe FUN-1 monomer into CIVS and an trations of the local anesthetics, with more than increase in diffuse intracellular fluorescence, as 90% ofthe intracellularcation beinglostduringthe demonstrated by flowcytomctry. These alterations initial 10 minutes after exposure to 0.3, 50, or 30 were already detectable after an incubation period mg/mL ofbenzydamine, lidocaine, or bupivacaine, 132 INFEC770USDISEASESINOBSTETRICSANDGYNECOLOGY LOCALANESTHETICSACTIVITYAGAINSTCANDIDA PINA-VAZETAL. Fig. 6. /,. Untreated spheroplasts of C. albicans, ATCC strain 10231. Notice the loose partially digested cell walls (W), the intact protoplastwith nucleus, mitochondria, and vacuoles. Section stained with uranyl-lead. (x 9600). B. Spheroplast from the same sample as in Fig. 6A. Section treatedwith5%hydrogen peroxidefor 10minutesfollowed by lead staining.2s Notice the abundant ribosomes in the cytoplasmic matrixand 3 mitochondria (M). (x 50,600). C. Spheroplasts from the same preparation as in Fig. 6A but exposed to 0.3 mg/mL ofbenzydamine for 15 min. Notice several lysed spheroplasts (*) with collapsed cell walls. The nonlysed spheroplasts have a very altered ultrastructure. Section stained with uranyl-lead. (x 16,000). respectively. This indicates that the fungicidal ef- roplastsinanosmoticallyprotectivemedium,when fect results from a direct damage to the cell mem- exposed to fungicidal concentrations ofthe drugs. brane, rather than from a metabolic impairment Bacterialprotoplasts are quicklylysed bytreatment leading to secondary membrane damages. In sup- with phenethyl alcohol, tetrazolium salts, or local port of this interpretation is our observation that anesthetics, molecules that are known to directly exposure ofthe Candidacells to fungicidal concen- disorganize bacterial cell membranes.9’es The ul- trations ofthe metabolic inhibitor sodium azide in- trastructural studies revealed a second support for duced K leakage at a much slower rate as com- the assumption, which was the severe membrane pared to the three local anesthetics. As expected, alterations, with fracturing and solubilization, seen the membrane-active antimicrobial agent ampho- after 10 minutes ofexposure to fungicidal concen- tericin B also induced an extensive and rapid K trations ofthe drugs. leakage. The extensive loss ofintracellular K may The poorcorrelation found betweenthe MIC or not bc the sole factor responsible forthe fungicidal LC.s0 of bcnzydamine and those of lidocaine and activity of the drugs, as the membrane disorgani- bupivacaine suggests that although the drugs kill zation resulted in multiple perturbations thateven- Candida by acting on a common target (the cell tually could be lethal. membrane), the mechanisms for the membrane Additional support for a direct membrane dam- damage would differ in regards to benzydaminc on aging action offungicidal concentrations ofthe an- the one hand and lidocainc and bupivacaine on the esthetics was evident from the quick lysis ofsphe- other. INFECTIOUSDISEASESINOBSTETRICSANDGYNECOLOGY 133