Journal of Antimicrobial Chemotherapy (2003) 51, 281–287

DOI: 10.1093/jac/dkg088
Advance Access publication 14 January 2003

Treatment of plastic and extracellular matrix components with

chlorhexidine or benzalkonium chloride: effect on Candida albicans
adherence capacity in vitro
Christine Imbert*, Edith Lassy, Gyslaine Daniault, Jean-Louis Jacquemin and Marie-Hélène Rodier

Laboratoire de Parasitologie et Mycologie Médicales, Centre Hospitalier Universitaire La Milétrie, BP 577,

86021 Poitiers Cedex, France

Received 2 May 2002; returned 11 September 2002; revised 10 October 2002; accepted 19 November 2002

This study investigates the influence of treatment of plastic and extracellular matrix (ECM) pro-
teins with chlorhexidine or benzalkonium chloride on subsequent adherence of Candida albicans.
Three concentrations were tested for each antiseptic: (i) chlorhexidine, MIC (6.25–12.5 mg/L),
80 × MIC and 800 × MIC; and (ii) benzalkonium chloride, MIC (3.12 mg/L), 40 × MIC and 1600 × MIC.
Chlorhexidine and benzalkonium chloride activities were correlated with the tested concentra-
tions. Antiseptics used at MIC were unable to modify the adherence to plastic or ECM proteins.
Chlorhexidine (80 × MIC) induced a decrease in plastic adherence of 31% of the 15 strains used
and an increase in ECM protein adherence of 13% of strains. Benzalkonium chloride (40 × MIC)
induced a decrease in adherence to ECM proteins or plastic of 13–27% of strains. Our results
indicated that the treatment with 1600 × MIC benzalkonium chloride could induce the opposite
effect on adherence, depending on the surface: 60% of the strains showed an increase in their
adherence to ECM proteins, whereas 93% of the strains showed a decrease in their adherence
to plastic. A similar phenomenon was observed after treatment with 800 × MIC chlorhexidine:
60% of the strains showed an increase in their adherence to ECM proteins, whereas 67% showed
a decrease in adherence to plastic. Treatment of medical devices with at least 5000 mg/L of
chlorhexidine or benzalkonium chloride could therefore reduce C. albicans adherence to plastic
surfaces, but would be unable to prevent fungal adherence to ECM proteins.

Keywords: Candida, adherence, antiseptic agents

Introduction the increasing use of intravenous catheters.3 Infections associ-

ated with the use of central venous catheters (CVCs) can
Candida species are increasingly important nosocomial result in serious medical complications and expensive care,
pathogens and Candida albicans remains the most frequently and this is the most frequent factor limiting their prolonged
reported, even if other species of Candida are reported with an use.4,5 Infectious complications remain frequent for all types
increased frequency.1 According to a widespread American of CVC.6
study, the rate of invasive fungal infections among hospital Antiseptics are used to clean and disinfect traumatic
patients approximately doubled between 1980 and 1990, and wounds, mucous membranes, hands, operation sites and some-
the incidence of nosocomial candidaemia alone increased times to impregnate catheters.7–11 The increased number of
five-fold.2 There are multiple reasons for the increase in C. albicans infections among denture wearers also calls for
incidence of invasive fungal infections in patients, including effective prevention, and numerous authors are interested in
intensity of chemotherapy employed in cancer patients, the evaluating the efficacy of surface antiseptics or those impreg-
prolonged use of immunosuppressive agents in bone marrow nated into various denture materials.12–15 In vitro studies have
transplant patients, the use of broad-spectrum antibiotics and suggested that catheters, denture materials and other non-
..................................................................................................................................................................................................................................................................

*Corresponding author. Tel: +33-5-4944-3959; Fax: +33-5-4944-3908; E-mail: Christine.Imbert@univ-poitiers.fr

...................................................................................................................................................................................................................................................................

281
© 2003 The British Society for Antimicrobial Chemotherapy
 C. Imbert et al.
biological materials impregnated with some antiseptics, anti-
biotics or silver could resist infection more efficiently.12,14,16–20
In the present study, chlorhexidine gluconate and benzal-
konium chloride were chosen as the candidate drugs. The aim
of this work was to evaluate and compare the in vitro effect of
a pre-treatment of two different surfaces—untreated plastic
or plastic overlaid with extracellular matrix (ECM) proteins
—with chlorhexidine or benzalkonium chloride on subse-
quent infection by C. albicans isolates. The concentrations
used corresponded to: (i) MIC; (ii) concentrations usually
used on mucosa; and (iii) concentrations usually used on
plastic surfaces.
Materials and methods
Organisms and growth conditions
Fifteen isolates of C. albicans were studied; they were all
isolated in our laboratory from patients with septicaemia. The
identification of these clinical isolates was carried out by
using conventional physiological and morphological studies
such as the germ-tube test in serum, agglutination (Bichro-
Latex, Fumouze, Levallois Perret, France) and metabolic
properties (API 20C, bioMérieux, Marcy-L’Étoile, France).
Yeasts were first grown for 48 h at 28°C on Sabouraud agar
slants (Sanofi Diagnostics Pasteur, Marnes-La-Coquette,
France), to obtain a fresh culture of synchronous stationary
yeast-phase C. albicans.21 A loopful of this culture was trans-
ferred to 25 mL of Yeast Nitrogen Base medium (YNB, Difco,
Detroit, MI, USA), supplemented with 300 mM galactose
(Sigma, St Louis, MO, USA; YNB-gal) and incubated for
36 h at 37°C, without shaking. Galactose promotes adherence
of C. albicans yeasts to cellular and plastic surfaces.22,23
Before use in the adherence experiments, blastospores were
harvested, washed twice in 0.1 M phosphate-buffered saline
(PBS, pH 7.2; bioMérieux) and adjusted to 1.5 × 107 blasto-
spores/mL.
MICs of antiseptics
Chlorhexidine digluconate 20% (Sigma) and benzalkonium
chloride (Sigma) were used. The MICs of chlorhexidine and
benzalkonium chloride were assessed by a broth dilution
method using RPMI-1640 medium with L-glutamine but
without bicarbonate, buffered with 0.165 M MOPS at pH 7.
Chlorhexidine and benzalkonium chloride were prepared as
stock solutions of 5000 mg/L in sterile water and serial dilu-
tions of antiseptics (50–0.024 mg/L) were prepared in RPMI
medium. Yeast inocula were prepared by suspension in RPMI
medium, adjusted to a final concentration of 104 yeasts/mL
and the MICs of each of the antiseptic agents were determined
after incubation for 48 h at 37°C, without shaking. The MIC
was defined as the lowest drug concentration showing no
282
visible fungal growth. All tests were carried out on two occa-
sions.
Immobilized ECM proteins
Extracellular matrix gel (ECM gel, Sigma) was coated on to
wells of 96-well tissue culture plates (polystyrene, Evergreen
Scientific, Los Angeles, CA, USA) according to the manu-
facturer’s instructions. This gel was composed primarily of
laminin, collagen type IV, heparan sulphate proteoglycan and
entactin.
Treatment of plastic and biological surfaces with
antiseptics
The culture plates of ECM gel-coated and uncoated poly-
styrene were treated for 5 min with different concentrations of
chlorhexidine or benzalkonium chloride. The test concen-
trations of chlorhexidine corresponded to the MIC, 80 × MIC
and 800 × MIC for each isolate of C. albicans, and the test
concentrations of benzalkonium chloride corresponded to the
MIC, 40 × MIC and 1600 × MIC for each isolate. These con-
centrations were selected to correspond with those usually
used on mucosa (chlorhexidine 500–1000 mg/L and benzal-
konium chloride 125 mg/L) or plastic surfaces (chlorhexidine
and benzalkonium chloride 5000 mg/L). The MICs of anti-
septics were also determined.
Adherence of C. albicans to polystyrene
Adherence experiments were carried out in untreated 96-well
tissue culture plates as described previously.21 Tetrazolium
salt XTT was used to assess the adherence of C. albicans
blastospores to wells of tissue culture plates: the principle was
based upon the reduction of XTT tetrazolium to tetrazolium
formazan by mitochondrially active C. albicans blastospores
in the presence of an electron-coupling agent, menadione.
Briefly, C. albicans blastospores were added to 96-well tissue
culture plates at an inoculum of 1.5 × 107 cells/mL in 150 µL
of PBS and were allowed to adhere to the polystyrene for 2 h at
37°C; half of the wells were then washed twice with PBS to
remove the non-adherent yeasts. Thereafter, 300 mg/L XTT
(Sigma) and 0.13 mM menadione (Sigma) were added to all
wells. Plates were incubated for 3 h at 37°C without shaking,
then gently agitated and XTT formazan measured at A492nm
(LP400 micro-plate reader, Sanofi Diagnostics Pasteur) in
washed and unwashed wells. The % adherence capacity of
each isolate was calculated as a mean of absorbance units in
washed wells/absorbance units in unwashed wells.
Background formazan values were determined with plates
that contained PBS only or PBS, XTT and menadione; these
values did not exceed 0.005 absorbance units and therefore
were not significant. All experiments were carried out twice
with six replicates.
 Candida albicans and antiseptics
Adherence of C. albicans to ECM proteins
Adherence of C. albicans to ECM proteins was measured
following a 3 h incubation with XTT and menadione, as out-
lined above.
Statistical analyses
An analysis of variance (ANOVA) and a Scheffé’s test were
conducted to determine differences among the test groups
(P < 0.05).
Results
MICs of antiseptics
Results showed that MICs of chlorhexidine for the 15 isolates
of C. albicans, obtained in RPMI broth, ranged between 6.25
and 12.5 mg/L, and the MIC of benzalkonium chloride was
3.12 mg/L in all cases.
Plastic surfaces treated with chlorhexidine
Effect on the adherence capacity to plastic. The influence of
the treatment of plastic with chlorhexidine on the adherence
capacity of C. albicans was dependent on the antiseptic
concentration. Chlorhexidine used at its MIC had no sig-
nificant effect. Chlorhexidine at 80 × MIC induced a decrease
(P ≤ 0.001) in the adherence capacity of five strains (31%)
compared with the control or with MIC chlorhexidine treat-
Figure 1. Effect of the treatment of plastic with chlorhexidine on the adherence of C. albicans. These data represent the average and standard
deviation for two experiments carried out with six replicates.
283
ment (Figure 1). Chlorhexidine at 800 × MIC was the most
effective concentration to modify adherence: 10 strains
(67%) were less adherent (P ≤ 0.001) than after no treatment,
nine strains (60%) were less adherent (P ≤ 0.001) than after
MIC treatment and four strains (27%) were less adherent
(P ≤ 0.001) than after the use of 80 × MIC chlorhexidine.
Effect on the adherence capacity to ECM proteins. The influ-
ence of the treatment of ECM components with chlorhexidine
was dependent on the antiseptic concentration, and the chlor-
hexidine used at its MIC had no significant effect (Figure 2).
Chlorhexidine at 80 × MIC induced an increase (P ≤ 0.001) in
the adherence capacity of two strains (13%) compared with
the control and of four strains (27%) compared with MIC
chlorhexidine treatment (Figure 2). Chlorhexidine at 800 ×
MIC had the greatest effect on adherence: nine strains (60%)
were more adherent (P ≤ 0.001) than without treatment, 10
strains (67%) were more adherent (P ≤ 0.001) than after MIC
treatment and seven strains (47%) were more adherent than
after 80 × MIC treatment.
Plastic surfaces treated with benzalkonium chloride
Effect on the adherence capacity to plastic. The effect on
adherence capacity of treatment of a plastic with benzal-
konium chloride was dependent on the antiseptic concen-
tration. The use of benzalkonium chloride at its MIC did
not induce any modification of the adherence capacity but the
use of benzalkonium chloride at a concentration of 40 × MIC
(125 mg/L) inhibited the adherence to plastic (P ≤ 0.001) of
 C. Imbert et al.

Figure 2. Effect of the treatment with chlorhexidine of plastic coated with ECM components on the adherence of C. albicans. These data represent
the average and standard deviation for two experiments carried out with six replicates.

Figure 3. Effect of the treatment of plastic with benzalkonium chloride on the adherence of C. albicans. These data represent the average and
standard deviation for two experiments carried out with six replicates.

four strains (27%) compared with control, and of three strains
(20%) compared with MIC treatment (Figure 3).
Yeasts were significantly less adherent to plastic (P ≤ 0.001)
when it was pre-treated with the highest tested concentration
of benzalkonium chloride (1600 × MIC, 5000 mg/L); 14
strains (93%) showed a decrease in adherence capacity com-
pared with control or with results obtained after treatment
with benzalkonium chloride at its MIC (Figure 3).
Our results also showed that 10 strains (67%) were less ad-
herent (P ≤ 0.001) after treatment of plastic with 1600 × MIC
benzalkonium chloride than after treatment with 40 × MIC
benzalkonium chloride (Figure 3).

284
 Candida albicans and antiseptics
Figure 4. Effect of the treatment with benzalkonium chloride of plastic coated with ECM components on the adherence of C. albicans. These data
represent the average and standard deviation for two experiments carried out with six replicates.
Effect on the adherence capacity to ECM proteins. The influ-
ence of treatment of ECM components with benzalkonium
chloride was dependent on the antiseptic concentration. The
chlorhexidine used at its MIC (3.12 mg/L) modified adher-
ence (P ≤ 0.001) of three strains (20%); the adherence of strain
182 was increased, whereas it was inhibited for strains 43 and
253. The use of benzalkonium chloride at a concentration of
40 × MIC (125 mg/L) inhibited adherence to ECM com-
ponents (P ≤ 0.001) of two strains (13%) compared with con-
trol or MIC treatment (Figure 4). The use of benzalkonium
chloride at a concentration of 1600 × MIC (5000 mg/L)
increased adherence of nine strains (60%) compared with
control, of 11 strains (73%) compared with MIC treatment
and of nine strains (60%) compared with 40 × MIC (Figure 4).
Discussion
Binding antiseptic substances to a catheter surface or to any
implanted medical prosthesis is one possible strategy to
overcome the problem of infections associated with these
implanted devices. We investigated the efficiency of two
major antiseptics, chlorhexidine and benzalkonium chloride,
on adherence of C. albicans blastospores after the pre-
treatment of plastic surfaces and ECM components.
Adherence is a prerequisite for C. albicans colonization
and is considered as an initial step in the process leading to
infection. When a non-biological material is implanted, there
is an initial attachment and a biofilm layer rapidly forms on its
surface. This biofilm is a monolayer or multilayer of cells
285
embedded within an ECM material. Biofilm microorganisms
are known to be resistant to host defence mechanisms and
antibiotic therapy, and can be released from the layer into the
surroundings, thus sustaining the infection.24 There is con-
siderable interest in understanding the mechanisms involved
in biofilm formation on catheters, which could lead to control
and intervention strategies for the prevention of systemic
candidiasis. Some authors have, for example, recently sug-
gested that filamentation promoted the formation and persist-
ence of cell populations on the biofilm that overlaid the
catheter.25
We have observed that the influences of chlorhexidine and
benzalkonium chloride on C. albicans adherence capacity
were correlated with the antiseptic concentration used to treat
the surfaces. These antiseptics, used at their corresponding
MIC, were unable to modify adherence to plastic or ECM
proteins efficiently. This result corroborated the recommen-
dations established for the use of antiseptics in the treatment
of plastic surfaces and mucosa, i.e. concentrations corres-
ponding to the MIC were not enough to prevent colonization.
The higher concentrations of chlorhexidine (800 × MIC)
and benzalkonium chloride (1600 × MIC) have been chosen
to correspond with those usually used to treat hard surfaces.
Our results indicated that surface treatment with 1600 × MIC
of benzalkonium chloride could enhance fungal adherence,
depending on the nature of the surface; 60% of the strains
showed a significant increase in their adherence capacity to
ECM proteins, whereas 93% of the strains showed a signifi-
cant decrease in their adherence capacity to plastic after treat-
ment. A similar phenomenon was observed after treatment of
 C. Imbert et al.
the tested surface with chlorhexidine at 800 × MIC; 60% of
the strains showed an increase in adherence to ECM proteins,
whereas 67% of the strains showed a decrease in adherence to
plastic after treatment. This phenomenon appeared as soon as
the chlorhexidine concentration reached 80 × MIC but was
not observed with benzalkonium chloride at 40 × MIC.
Benzalkonium chloride (40 × MIC) induced a decrease in
adherence to biological or plastic surfaces of 13–27% of the
strains, whereas an enhanced adherence was never observed.
The benzalkonium chloride concentration of 40 × MIC and
the chlorhexidine concentration of 80 × MIC were chosen to
correspond to those usually used to treat mucous surfaces, and
these results confirmed that they were unable to reduce fungal
adherence. The highest concentrations of chlorhexidine and
benzalkonium chloride that were tested were very efficient in
preventing C. albicans attachment to plastic. This is in agree-
ment with previous studies which suggested that catheters
impregnated with antiseptics exhibited reduced microbial
adherence compared with conventional catheters.16,18,19,26,27
However, our results also suggested that antiseptic pre-
treatment could promote adherence to ECM components.
C. albicans is able in vivo to adhere to a plastic device and then
to form a biofilm. Our results were obtained in vitro, but raise
questions about the benefit of treating medical devices with
antiseptic in the prevention of candidiasis if we only consider
the step of biofilm development. Cell surface hydrophobicity
plays a major role in adherence of C. albicans to plastic,
whereas adherence to ECM proteins involves specific
adhesins present at the surface of the fungal cell.28 The pro-
cesses implicated in adherence to a plastic or biological
surface are very different and this could explain why we
observed opposing influences of antiseptic treatments.
Our results correlate with other studies which suggested
that chlorhexidine is effective in preventing C. albicans
plastic attachment and growth on acrylic resin.14 McCourtie
et al.29,30 suggested that the pre-treatment of denture acrylic
with chlorhexidine gluconate reduced the subsequent adher-
ence of C. albicans. More recently, Ivanovski et al.17 have
evaluated the efficiency of disinfecting solutions incorpo-
rated into dental stone casts against one strain of C. albicans
and suggested that chlorhexidine was unable to achieve a
satisfactory level of disinfection. Other authors have shown
that chlorhexidine is an effective antifungal agent in vitro.31,32
It is nevertheless difficult to compare these results because
of the varying experimental conditions. Each study has been
carried out with only a few strains of C. albicans, and the test
concentrations of the antiseptics were often different.
To summarize, our results indicate that chlorhexidine and
benzalkonium chloride had opposing effects on the in vitro
adherence capacity of C. albicans depending on the nature of
the surface; adherence was inhibited on plastic surfaces and
increased on ECM proteins. This study also suggests that chlor-
hexidine and benzalkonium chloride have a comparable effect
286
on modifying fungal adherence to ECM components when
these antiseptics were used between 5000 and 10 000 mg/L.
However, these concentrations of benzalkonium chloride
were more efficient than chlorhexidine in preventing adher-
ence of the yeast cells to plastic surfaces.
Acknowledgements
We would like to acknowledge Dr S. Ragot for help with the
statistical analysis, and Dr P. Johnson for reviewing the
manuscript prior to submission.
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