Antifungal Activity of Local Anesthetics Against Candida Species

14 Pages·2012·5.81 MB·English

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