Synergistic Fungistatic Effects of Lactoferrin in Combination with Antifungal Drugs against Clinical Candida Isolates.PDF

A NTIMICROBIAL A GENTS AND C HEMOTHERAPY ,

0066-4804/99/$04.00

Nov. 1999, p. 2635–2641

Vol. 43, No. 11

Synergistic Fungistatic Effects of Lactoferrin in Combination

with Antifungal Drugs against Clinical Candida Isolates

M. E. KUIPERS, 1 *H.G. DE VRIES, 2 M. C. EIKELBOOM, 1 D. K. F. MEIJER, 1 AND P. J. SWART 1 †

Section of Pharmacokinetics and Drug Delivery, Groningen University Institute for Drug Studies, University Centre for

Pharmacy, 9713 AV Groningen, 1 and Section of Medical Microbiology, University Hospital Groningen,

9713 GZ Groningen, 2

The Netherlands

Received 9 November 1998/Returned for modiﬁcation 7 January 1999/Accepted 3 August 1999

ml 2 1 . Interestingly, in the combination

experiments we observed pronounced cooperative activity against the growth of Candida by using lactoferrin

and the three antifungals tested. Only in a limited concentration range was minor antagonism detected. The

use of lactoferrin and ﬂuconazole appeared to be the most successful combination. Signiﬁcant reductions in the

minimal effective concentrations of ﬂuconazole were found when it was combined with a relatively low

lactoferrin concentration (1 mg/ml). Such combinations still resulted in complete growth inhibition, while

synergy of up to 50% against several Candida species was observed. It is concluded that the combined use of

lactoferrin and antifungals against severe infections with Candida is an attractive therapeutic option. Since

ﬂuconazole-resistant Candida species have frequently been reported, especially in HIV-infected patients, the

addition of lactoferrin to the existing ﬂuconazole therapy could postpone the occurrence of species resistance

against ﬂuconazole. Clinical studies to further elucidate the potential utility of this combination therapy have

been initiated.

Since its discovery in 1839 by Langenbeck (16), the genus

Candida has been shown to be the causative agent of many

infections in an increasing range of anatomical sites and clin-

ical settings. In normal healthy individuals, the yeast Candida

is classiﬁed as a commensal organism that can colonize both

internal and external surfaces. Under these circumstances, an

equilibrium between the host and the yeast microﬂora ensures

the avirulent, commensal status of this microorganism. This

equilibrium is attained not only by speciﬁc immune responses

but also by nonspeciﬁc factors that are secreted in saliva or

mucosal secretions, such as immunoglobulin A, lysozyme, lac-

toferrin, and histatins (25, 27, 31). A range of predispositions,

of which the most common is an immunocompromised status

of the patient, can affect the equilibrium. For example the

onset of candidosis in people infected with human immunode-

ﬁciency virus (HIV) is considered to be closely related to

manifestation of AIDS and AIDS-related diseases. In fact, it

represents one of the factors that is used by the Centers for

Disease Control and Prevention to deﬁne AIDS (7). Although

Candida albicans has been implicated in the early stages of

AIDS, infections due to C. glabrata and C. krusei are becoming

more widespread in late-stage AIDS (13).

Before the emergence of the HIV epidemic, oral mycotic

infections were treated with polyene antifungals, such as am-

photericin B or nystatin, and with azoles, such as clotrimazole

or miconazole. The high relapse rate in HIV-positive patients

and reported toxic side effects of amphotericin B led to the use

of azoles as the ﬁrst line of treatment (10, 17). Because keto-

conazole and itraconazole are not as readily absorbed, their

use has been limited. Although ﬂuconazole has become the

agent of choice in antimycotic therapy, its widespread use has

resulted in an increase in resistance of Candida to its antifungal

efﬁcacy (1, 30). In addition, the use of 5-ﬂuorocytosine as

antimycotic has led to resistant strains of Candida being clin-

ically signiﬁcant as well (14). Because of the rising incidence of

failures in the treatment of mycoses in the case of severely

immunosuppressed patients, there is a need for the develop-

ment of new therapeutic agents that support the antifungal

activity of antimycotics (41).

As mentioned, saliva contains several nonspeciﬁc defense

factors. Among them is lactoferrin, an iron-binding glycopro-

tein present at relatively high concentrations (up to 12 mg/ml)

in many exocrine ﬂuids (33). The protein has broad-spectrum

antimicrobial properties against bacteria, yeasts, and viruses

and is considered to play an important role in the host defense

against infections on mucosal surfaces and in colostrum and

milk (4, 6, 12, 15, 33, 37, 38).

The anti- Candida activity of lactoferrin, ﬁrst reported by

Kirkpatrick et al. (15), is commonly attributed to its ability to

bind and sequester environmental iron (6, 33). Yet the iron-

free form of lactoferrin (apo-lactoferrin) is able to kill both C.

albicans and C. krusei , by mechanisms related to alterations in

cell surface permeability (24, 37). Moreover, lactoferricin B, a

* Corresponding author. Present address: Yamanouchi Europe BV,

Clinical Pharmacology Research Department, Elisabethhof 1, 2353

EW Leiderdorp, The Netherlands. Phone: 31-71-5455283. Fax: 31-71-

5455276. E-mail: kuipers.nl@yamanouchi-eu.com.

† Present address: Yamanouchi Europe BV, Bioanalysis and Drug

Metabolism Section, 2353 EW Leiderdorp, The Netherlands.

2635

Because of the rising incidence of failures in the treatment of oropharyngeal candidosis in the case of

severely immunosuppressed patients (mostly human immunodeﬁciency virus [HIV]-infected patients), there is

need for the development of new, more effective agents and/or compounds that support the activity of the

common antifungal agents. Since lactoferrin is one of the nonspeciﬁc host defense factors present in saliva that

exhibit antifungal activity, we studied the antifungal effects of human, bovine, and iron-depleted lactoferrin in

combination with ﬂuconazole, amphotericin B, and 5-ﬂuorocytosine in vitro against clinical isolates of Candida

species. Distinct antifungal activities of lactoferrin were observed against clinical isolates of Candida . The

MICs generally were determined to be in the range of 0.5 to 100 mg

2636

KUIPERS ET AL.

A NTIMICROB .A GENTS C HEMOTHER .

peptide produced by enzymatic cleavage of bovine lactoferrin,

was found to have a lethal effect on Candida species through

direct interaction with the cell surface (3).

The need for reducing the development of microbial resis-

tance has led to experiments in which the cooperative effects of

lactoferrin and currently used treatments were assessed. This

resulted in an increased resistance to apo-lactoferrin-mediated

cell death when physiological concentrations of apo-lactoferrin

and sub-MICs (concentrations causing substantial but incom-

plete growth inhibition) of antifungal drugs or agents were

tested (23). However, others showed that combining lactofer-

rin and several antifungals led to a decrease in the concentra-

tion of lactoferrin required to inhibit the growth of Candida ,

whereas the combination of lactoferrin and clomitrazole even

resulted in synergistic anti- Candida activity (41). In the present

study we investigated in vitro the antifungal effect of lactoferrin

combined with some common antifungal agents against several

clinical isolates of Candida . Potential cooperative or synergistic

anti- Candida activity with lactoferrin and antifungals would

enable a lower dose of standard antimycotics during antimy-

cotic therapy and might at the same time decrease the induc-

tion of drug-resistant species.

microplate reader (El X 800; Bio-Tek Instruments, Inc., Winooski, Vt.) after re-

suspension of the contents of the wells with a multichannel pipette. Any bubbles

were removed with the tip of a sterile needle. For any wells not producing a visual

or spectrophotometrically measurable increase in turbidity after 48 h, the ab-

sence of growth was conﬁrmed by inoculating 20

l of the well contents onto

SDA and then incubating for 5 days at 35°C in air.

The MIC was deﬁned as the lowest concentration of antifungal agent that

substantially inhibited the growth of Candida after 24 h, according to the rec-

ommendations of the National Committee for Clinical Laboratory Standards

(NCCLS) for the antifungal agents used (22). All experiments were performed in

quadruplicate.

Synergy experiments. The combined effects of bovine lactoferrin and ﬂucon-

azole, amphotericin B, or 5-ﬂuorocytosine against the growth of Candida species

were examined under the same experimental conditions used for determination

of the MICs. A dilution matrix (eight by eight) with eightfold drug dilutions,

which also included the drugs used individually, was prepared. The results of the

turbidity measurements at 630 nm were used to calculate the inhibiting effects of

the drug combinations on Candida growth. Maximum Candida growth was set at

0%, and complete inhibition of Candida growth was set at 100%. The results

obtained are presented as growth inhibition curves. In addition, for character-

ization of drug-drug interactions, a three-dimensional analytical method was

used according to the method described by Prichard and Shipman (32). This

experimental design was used to identify regions at which signiﬁcant drug inter-

actions occurred. In brief, theoretical additive interactions were calculated from

the dose-response curves of the individual drugs. The theoretical calculated

additive interactions, representing the predicted additive interactions, were then

subtracted from the experimental interactions, obtained via the effects of the

drug combination on the turbidity measurements at 630 nm, to reveal dose-

response ranges of greater-than-expected interactions (synergy). The resulting

surface would appear as a horizontal plane at 0% inhibition above the calculated

additive surface if the interactions were merely additive. Any peaks above this

plane would be indicative of synergy. Similarly, any depressions below the plane

would indicate antagonism. For example, a combination of 0.5 mg of lactoferrin

per ml and 100

MATERIALS AND METHODS

Microorganisms. Three C. albicans strains, four C. glabrata strains, and one C.

tropicalis strain, isolated from the oral cavity and differing in their susceptibilities

to the antifungal agents amphotericin B, ﬂuconazole, and 5-ﬂurocytocine, were

obtained from the routine microbiology laboratory of the University Hospital

Groningen, Groningen, The Netherlands. C. albicans ATCC 10231 was used as

a control in all susceptibility tests. All strains were stored on Sabouraud dextrose

agar (SDA) slopes (Oxoid, Unipath Ltd., Basingstoke, United Kingdom) at 4°C.

Assay media. For the experiments we used Sabouraud liquid medium (SLM)

(pH 5.6) (Oxoid, Unipath Ltd.) and RPMI 1640 medium (with L -glutamine,

without NaHCO 3 , and supplemented with 2% glucose; pH 7.0) (Gibco BRL,

Paisley, Scotland). These media were prepared according to the manufacturer’s

instructions. RPMI 1640 was used when the antifungal activity of 5-ﬂuorocy-

tosine was studied. All other compounds were assayed in SLM.

Antifungal agents. Bovine lactoferrin and human lactoferrin (both from Nu-

mico Research B.V., Wageningen, The Netherlands) were dissolved in assay

medium at appropriate concentrations. Iron-free lactoferrin (apo-lactoferrin)

was prepared from bovine lactoferrin by overnight dialysis against 0.1 M citric

acid by the method earlier described by Masson et al. (19) and dissolved in assay

medium at appropriate concentrations. Fluconazole (Diﬂucan I.V.; Pﬁzer B.V.,

Capelle aan den Yssel, The Netherlands) and 5-ﬂuorocytosine (Ancotil; Roche

Nederland B.V., Mijdrecht, The Netherlands) were dissolved in assay medium at

appropriate concentrations. Amphotericin B (Fungizone; Bristol-Myers Squibb

Company, Woerden, The Netherlands) was prepared to a concentration of 5

mg/ml in sterile water and was further diluted in assay medium. All suspensions

were prepared in sterile glass tubes before addition to the microtiter plate.

In order to exclude the antifungal activity of endotoxins present in the lacto-

ferrin preparations, the anti- Candida effect of lipopolysaccharide (LPS) (Bio-

whittaker, Inc. Walkerville, Md.) alone was tested in a range of 1 to 1,000 pg/ml

in our assay system as well.

Inoculum. The yeast isolates were grown on SDA for 24 h at 35°C in air.

Suspensions were made by isolating ﬁve colonies from these cultures. These were

suspended in 10 ml of SLM and mixed while being incubated for 18 h at 35°C in

air. From this culture, a 1:10 dilution in SLM was incubated for 5 h, resulting in

a culture in its growth phase. This was vortex mixed, and the turbidity was

adjusted to the density of a 0.5 McFarland barium sulfate turbidity standard at

530 nm, resulting in a concentration of 1

50% would

indicate that the speciﬁed drug combination in this speciﬁed concentration range

would cause a 50% synergistic antifungal effect. In other words, the growth of the

Candida isolate is inhibited more efﬁciently (namely, 50% more efﬁciently) with

the speciﬁed drug combination. Likewise, a peak of

g of ﬂuconazole per ml resulting in a peak of

25% would indicate an

antagonistic antifungal effect of the speciﬁed drug combination, meaning that the

growth of the Candida isolate is inhibited less than was expected on the basis of

the individual dose-response curves (namely, 25% less efﬁciently).

RESULTS

10 6 cells per ml as

previously described (29). From this, the test inoculum was prepared to a con-

centration of 1

10 6 to 5

Inhibition of Candida growth. The activities of various forms

of lactoferrin (bovine and human lactoferrins and bovine apo-

lactoferrin) against several clinical isolates of C. albicans , C.

glabrata , and C. tropicalis were determined and compared to

the susceptibilities of Candida species to currently used anti-

mycotics. The MICs were determined, according to the recom-

mendations by the NCCLS (22), after 24 h of incubation of the

Candida species with the antifungal agents and are presented

in Table 1.

Since the antifungal activity of human lactoferrin was com-

parable to or even less than that of the bovine variant (results

not shown), we continued all other experiments with bovine

lactoferrin because of its better availability. Bovine lactoferrin

and bovine apo-lactoferrin exhibited equivalent antifungal ac-

tivities. The MICs found for these two variants were all in the

same range and for the Candida species tested in SLM medium

were in the range of 0.5 to 100 mg/ml (Table 1).

The antifungal activities of the other currently used antimy-

cotics in our test system were comparable to those obtained

earlier from the microbiology services of the University Hos-

pital, which supports the accuracy of our test system (results

not shown).

Because lactoferrin is able to bind LPS (8), we wanted to

exclude the contribution of LPS to the antifungal activity of

these milk proteins. We noted that at concentrations of up to

1,000 pg of LPS per ml no killing of Candida species occurred.

Cooperative or synergistic activity. From the panel of clin-

ical isolates we chose four Candida species, depending on their

susceptibility to the tested antifungals, to be examined for the

combined activity of lactoferrin and ﬂuconazole, amphotericin

10 4 cells per ml by a 1:100 dilution in SLM.

Conﬁrmation of the inoculum size was done by using a model C Spiral Plater

(Spiral Systems, Inc., Cincinnati, Ohio). One hundred microliters was automat-

ically plated out onto SDA and incubated for 18 h at 35°C in air, and the

concentration was calculated according to the manufacturer’s speciﬁcations.

Assay format. To each well of a sterile 96-well plastic ﬂat-bottom tray with

matching covers (Corning Costar, Cambridge, United Kingdom), 50

10 4 to 5

l of test

inoculum was added. Appropriate concentrations of the antifungal agents to be

tested were added to the wells (75 to 150

l). Controls were included for the

determination of the growth characteristics of each Candida species without the

presence of an antifungal agent. The ﬁnal volume per well was adjusted to 200

l with the assay medium used (SLM or RPMI).

Incubation, growth curves, and end point criteria. After inoculation, plates

were incubated for 24 h at 35°C in air without agitation. Turbidity measurements

were performed at 0 h, hourly at 18 to 24 h, and 48 h at 630 nm in an automated

V OL . 43, 1999

SYNERGISTIC EFFECTS OF LACTOFERRIN ON CANDIDA GROWTH

2637

TABLE 1. MICs of several antifungal agents against Candida species

MIC a

(mean

SD)

Isolate b

Species

Lactoferrin (bovine)

(mg/ml)

Apo-lactoferrin

(mg/ml)

Amphotericin B

(

Fluconazole

(

5-Fluorocytosine

(

g/ml)

10231

C. albicans

0.06

0.05

— d

—

Y098

C. albicans

0.08

0.03

—

Y106

C. albicans

0.5

0.08

0.03

—

Y127

C. albicans

0.2

0.07

—

Y110

C. glabrata

0.2

0.06

156

—

Y111

C. glabrata

0.1

0.07

—

Y112

C. glabrata

0.4

0.05

—

Y110 c

C. glabrata

—

0.03

0.005

Y140 c

C. tropicalis

—

a The MIC was deﬁned as the lowest concentration of the antifungal agent that substantially inhibited the growth of Candida after 24 h, according to the

recommendations of the NCCLS for the antifungal agents tested (22). All experiments were performed in quadruplicate.

b Isolate 10231 is an ATCC strain; all other isolates are clinical, mostly oral, Candida isolates.

c The isolate was tested in RPMI medium instead of SLM.

d —, not determined.

B, and 5-ﬂuorocytosine. For this we used a dilution matrix with

eight- by eightfold drug dilutions and we measured the effects

on Candida growth. The results are presented as calculations

of the effects of both compounds on the inhibition of Candida

species and also are represented by so-called synergy plots, in

which the actual experimental dose-response values were com-

pared with the theoretical dose-response values (see Materials

and Methods). For an additive interaction of the two antifun-

gals, the actual experimental dose-response curves should co-

incide with the theoretical ones (with no exclusion of Candida

growth inhibition), but any peaks above or below these values

(above or below baseline [0%]) would be indicative of syner-

gistic or unwanted antagonistic interactions, respectively.

Combination of ﬂuconazole and lactoferrin. The combined

effect of ﬂuconazole and lactoferrin against the growth of Can-

dida isolate Y110 is shown in Fig. 1. It was found that this

Candida strain was completely inhibited in its growth by using

g/ml and 31 mg/ml, respectively (Table 1). This implies

that a complete inhibition of growth is possible with lower

concentrations of antimycotic than could be extrapolated from

the MICs. This applies to other combinations of lactoferrin

and ﬂuconazole as well (Fig. 1). Several combinations of ﬂu-

conazole and lactoferrin resulted in a clear synergistic anti-

Candida effect against Y110, as shown in Fig. 2. Effects above

baseline from

50% were observed. For example, a

combination of 0.5 mg of lactoferrin per ml and 100

5to

gof

ﬂuconazole per ml resulted in 50% synergistic effects, whereas

25 mg of lactoferrin per ml in combination with 3.3

gof

ﬂuconazole per ml induced only a 5% extra anti- Candida ef-

fect. In the range of the MICs of both compounds, synergistic

effects were not evident. This was expected because concen-

trations of ﬂuconazole in the range of its MIC should be

capable in themselves of complete Candida growth inhibition.

Importantly, no antagonistic anti- Candida activity between lac-

toferrin and ﬂuconazole was observed for this isolate.

Y127, a rather lactoferrin-insensitive Candida isolate (MIC

of 98 mg/ml), was completely inhibited by using 10 mg of

g of ﬂuconazole per ml in combination with 10 mg of

lactoferrin per ml, whereas their MICs against this isolate were

FIG. 1. Combined inhibitory effects of lactoferrin and ﬂuconazole on the

growth of C. glabrata isolate Y110. Presented is the top elevation of a three-

dimensional dose-response graph (concentration of ﬂuconazole versus concen-

tration of lactoferrin versus percent growth inhibition). The amount of inhibition

of the Candida growth is indicated by the bar at the right.

FIG. 2. Combined inhibitory effects of lactoferrin and ﬂuconazole on the

growth of C. glabrata isolate Y110. The graph demonstrates the amount of

synergy (i.e., potentiation of inhibition above expected additivity) observed with

the combination of the two compounds. Presented is the front elevation of the

synergy plot. The amount of synergy is indicated by the bar at the right.

156

2638

KUIPERS ET AL.

A NTIMICROB .A GENTS C HEMOTHER .

FIG. 3. Combined inhibitory effects of lactoferrin and ﬂuconazole on the

growth of C. albicans isolate Y127. Presented is the top elevation of a three-

dimensional dose-response graph (see the legend to Fig. 1). The amount of

inhibition of the Candida growth is indicated by the bar at the right.

FIG. 5. Combined inhibitory effects of lactoferrin and amphotericin B on the

growth of C. glabrata isolate Y110. Presented is the top elevation of a three-

dimensional dose-response graph (see the legend to Fig. 1). The amount of

inhibition of the Candida growth is indicated by the bar at the right.

lactoferrin per ml in combination with 1

g of ﬂuconazole per

g/ml (Fig. 3). For

this species, antagonistic effects along with synergistic effects

on Candida growth inhibition were found, in the range of

g of ﬂuconazole per ml) resulted in a complete inhi-

bition of Candida growth. The cooperative activities of ﬂucon-

azole and lactoferrin tended to be slightly antagonistic (10%)

with minor amounts of both compounds (0.005 mg of lactofer-

rin per ml and 0.3

40% (20% antagonism to 40% synergism). For example, a

combination of 1 mg of lactoferrin per ml and 0.05

gof

ﬂuconazole per ml resulted in about 20% antagonistic effects;

in other words, 20% less inhibition of Candida growth was

found than theoretically could be expected on basis of the

individual inhibitory effects of lactoferrin and ﬂuconazole. In

contrast, 25 mg of lactoferrin per ml in combination with 0.5

g of ﬂuconazole per ml) but were clearly

synergistic (50%) with concentrations of 0.5 mg of lactoferrin

per ml and 10

g of ﬂuconazole per ml.

Combination of amphotericin B and lactoferrin. Efﬁcient

inhibition of the Candida growth was also observed with the

combination of amphotericin B and lactoferrin against isolate

Y110. A complete inhibition of the Candida growth was ob-

served with 0.5 mg of lactoferrin per ml and 0.1

g of ﬂuconazole per ml induced as much as 40% extra growth

inhibition (Fig. 4).

Likewise, Y111, one of the more lactoferrin-sensitive strains,

was efﬁciently inhibited by combinations of lactoferrin and

ﬂuconazole. A reduction of more than 50% of the MICs of

g of ampho-

tericin B per ml (Fig. 5). In addition, both moderate antago-

nistic (10%) and also synergistic (30%) inhibition of the

Candida growth was demonstrated with certain concentration

ranges (Fig. 6).

In the case of isolate Y127, the combination of lactoferrin

and amphotericin B did not result in a pronounced decrease in

the required concentrations of lactoferrin or amphotericin B to

obtain complete Candida inhibition. Complete inhibition of

the Candida growth could be obtained only with concentra-

tions of amphotericin B or lactoferrin close to their MICs. Yet,

slightly antagonistic (10%) as well as synergistic (30%) effects

of this combination against Y127 could be observed. The 30%

synergistic effects were detected with 30 mg of lactoferrin per

ml in combination with 0.1

g of amphotericin B per ml, which

is only a small decrease from the MIC of amphotericin B itself

(0.15

g/ml).

Combination of 5-ﬂuorocytosine and lactoferrin. A combi-

nation of 5-ﬂuorocytosine and lactoferrin resulted in an effec-

tive inhibition of the growth of isolate Y110. A 100% growth

inhibition was observed with 0.0008

g of 5-ﬂuorocytosine per

ml (a decrease of 30-fold compared to its established MIC) in

combination with 0.02 mg of lactoferrin per ml. This combina-

tion of antifungals did demonstrate a clear synergistic activity

against Y110 of 50% with 0.025

FIG. 4. Combined inhibitory effects of lactoferrin and ﬂuconazole on the

growth of C. albicans isolate Y127. The graph demonstrates the amount of

synergy (i.e., potentiation of inhibition above expected additivity) observed with

the combination of the two compounds. Presented is the front elevation of the

synergy plot. The amount of synergy is indicated by the bar at the right.

g of 5-ﬂuorocytosine per ml

in combination with 0.01 mg of lactoferrin per ml. Yet, minor

antagonistic effects of 5% on the growth inhibition were also

ml, while the MIC of ﬂuconazole was 10

both compounds against this isolate (1 mg of lactoferrin per ml

with 10

V OL . 43, 1999

SYNERGISTIC EFFECTS OF LACTOFERRIN ON CANDIDA GROWTH

2639

FIG. 6. Combined inhibitory effects of lactoferrin and amphotericin B on the

growth of C. glabrata isolate Y110. The graph demonstrates the amount of

synergy (i.e., potentiation of inhibition above expected additivity) observed with

the combination of the two compounds. Presented is the front elevation of the

synergy plot. The amount of synergy is indicated by the bar at the right.

fungal activities of the various lactoferrins and some common

antifungals alone. We found that lactoferrin is able to inhibit

the growth of several Candida species and that the MICs of this

milk protein commonly varied between 0.5 and 100 mg/ml. The

potential mechanisms by which lactoferrin inhibits the growth

of yeasts were discussed by Nikawa et al. (24) earlier. Struc-

tural changes in the microbial cell wall, indirect effects on

enzyme activation, an increased generation of metabolic by-

products of aerobic metabolism, iron deprivation, and combi-

nations of these factors were mentioned as possible explana-

tions for the fungicidal activity of lactoferrin. The differences in

activity against different Candida species detected in the

present study can be explained by an unequal effectiveness of

lactoferrin due to differences in cell wall composition, sensitiv-

ity for enzyme activation, or the need for iron in the distinct

Candida species (24).

Several studies demonstrated the antifungal activity of the

iron-free from of lactoferrin, apo-lactoferrin, whereas in the

same studies the native form of lactoferrin did not show any

effect on Candida growth inhibition (15, 24). However, in our

present study we showed that both apo-lactoferrin and lacto-

ferrin were able to inhibit the growth of several clinical isolates

of Candida , albeit to different extents. Since the potential

mechanism for the antifungal activity of lactoferrin, as well as

that of apo-lactoferrin, is not likely to be directed to a single

phenomenon (24), the observed differences in activity between

these two proteins can also be explained by unequal effectivi-

ties of the proteins against different Candida species, due to

differences in cell wall composition, sensitivity for enzyme ac-

tivation, or the need for iron in the distinct Candida species.

In an earlier experiment we determined the LPS content of

lactoferrin (5 pg/mg of protein). In our present experiment we

found that 1,000 pg of LPS per ml was not able to kill the

Candida species. Because concentrations of lactoferrin of up to

100 mg/ml do contain 500 pg of LPS per ml, we can therefore

assume that the milk protein itself predominantly causes the

inhibition of Candida species.

In the literature there is no consensus with regard to the

inhibitory potency of lactoferrin against Candida species. In-

hibition of Candida growth has been tested with protein con-

centrations of as low as 20

g of 5-ﬂuorocytosine per ml in combina-

tion with 0.01 mg of lactoferrin per ml.

The C. tropicalis isolate Y140 is a 5-ﬂuorocytosine-resistant

isolate (see also Table 1). We investigated antifungal effects

against Y140 with only a combination of 5-ﬂuorocytosine and

lactoferrin. Unfortunately, a complete inhibition of Candida

growth could not be observed; only 90% inhibition was

reached. In addition, only moderate synergistic effects of 15%

with 10 mg of lactoferrin per ml and 10

g of 5-ﬂuorocytosine

per ml and antagonistic effects of 10% with 0.001 mg of lacto-

ferrin per ml and 45

g of 5-ﬂuorocytosine per ml were seen.

DISCUSSION

C. albicans and other Candida species are common patho-

gens that frequently cause oral infections in immunocompetent

and immunocompromised individuals due to the suppression

of local as well as systemic defense mechanisms (34). Due to

the increased incidence of fungal infections, a collection of

broad-spectrum antifungal agents has been introduced. For

example, HIV-infected patients who undergo several episodes

of oral thrush have been extensively treated with ﬂuconazole,

one of the available triazoles. Yet, ﬂuconazole-resistant Can-

dida species have been observed even in patients who did not

have an azole treatment history. These patients were rather

severely immunocompromised (low CD4 count or C2 or -3

category of HIV infection) (39). An attractive therapeutic op-

tion in these circumstances might be a combination of active

agents with different modes of action.

Earlier studies already showed that intrinsic components of

the host defense system are capable of killing several Candida

species, such as histatins and several saliva proteins, like ly-

sozyme and lactoferrin. These products therefore represent

attractive choices to be used in combination with the common

antifungal drugs, since the availability and toxicity of the en-

dogenous agents are of no concern.

We focused our attention on lactoferrin, since it was dem-

onstrated that this milk protein not only is able to inhibit the

growth of several bacteria and Candida species but also has

important activity against several viruses (HIV, herpes simplex

virus, and human cytomegalovirus) (12, 18, 38).

Prior to the combination experiments, we tested the anti-

g/ml, while sub-MICs (concentra-

tions of antifungal agents causing substantial but incomplete

growth inhibition of Candida )of100

g/ml have been reported

(41). However, exact MICs often were not determined. In

addition, the varying experimental conditions, such as differ-

ences in medium composition, pH, incubation temperature,

incubation time, and end point criteria, as well as the variable

use of human versus bovine lactoferrin and of iron-free lacto-

ferrin (apo-lactoferrin) versus iron-containing lactoferrin,

make a useful comparison of the present results with those

reported earlier difﬁcult.

The multiple fungistatic mechanisms of lactoferrin make this

protein a promising compound for combination therapy. A

synergistic fungistatic activity of a combination of drugs can be

anticipated in particular when the drugs used have different

mechanisms of action. The drugs tested in combination with

lactoferrin in this study have distinct modes of activity. Flucon-

azole inhibits ergosterol synthesis by the inhibition of micro-

somal cytochrome P450. 5-Fluorocytosine is converted either

to 5-ﬂuorouridine triphosphate, a precursor for cellular RNA,

or to 5-ﬂuorodeoxyuridylic acid, a potent inhibitor of thymidy-

late synthase, both of which inhibit DNA synthesis by the

fungus. Amphotericin B, on the other hand, interacts with

ergosterol in the plasma membranes. In addition, recent pub-

lications suggested that the antifungal activity of amphotericin

B might also be mediated by oxidative damage (28, 36). At ﬁrst

observed with 0.001

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: