and other Candida
species are common pathogens 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 Candida
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 option 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 lysozyme 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 endogenous agents are of no concern.
We focused our attention on lactoferrin, since it was demonstrated 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
Prior to the combination experiments, we tested the antifungal 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. Structural changes in the microbial cell wall, indirect effects on enzyme activation, an increased generation of metabolic by-products of aerobic metabolism, iron deprivation, and combinations of these factors were mentioned as possible explanations 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, sensitivity for enzyme activation, or the need for iron in the distinct Candida
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
). However, in our present study we showed that both apo-lactoferrin and lactoferrin 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 effectivities of the proteins against different Candida
species, due to differences in cell wall composition, sensitivity for enzyme activation, or the need for iron in the distinct Candida
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. Inhibition of Candida
growth has been tested with protein concentrations of as low as 20 μg/ml, while sub-MICs (concentrations of antifungal agents causing substantial but incomplete growth inhibition of Candida
) of 100 μg/ml have been reported (41
). However, exact MICs often were not determined. In addition, the varying experimental conditions, such as differences 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 lactoferrin (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. Fluconazole inhibits ergosterol synthesis by the inhibition of microsomal 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 thymidylate 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 publications suggested that the antifungal activity of amphotericin B might also be mediated by oxidative damage (28
). At first sight, the interaction with ergosterol makes amphotericin B a less favorable candidate for combination with lactoferrin, which also interacts with the cell membrane. However, different sites of interaction at the membrane are possible and can even lead to additive effects.
In the present study we show a synergistic activity between lactoferrin and several antifungal agents. Even though the claim of synergism is definition dependent, it can be clearly stated that a substantial cooperative effect of lactoferrin with fluconazole, amphotericin B, and 5-fluorocytosine was observed. The combination of lactoferrin and fluconazole appeared to be the most successful combination. Significant reductions in necessary fluconazole concentrations with addition of lactoferrin still resulted in complete growth inhibition, and synergy of up to 50% against several Candida
species was noted. This result is in line with several studies that reported at least some synergistic antifungal activity of fluconazole and combinations of other compounds. Barchiesi et al. reported a twofold reduction in MICs by using a combination of terbinafine and fluconazole (2
). Scott et al. described synergistic antifungal activities with fluconazole and ibuprofen (35
). The combination of neutrophils or macrophages with fluconazole also showed a synergistic antifungal effect (5
). Also, the presence of even 5% human serum in the assay medium was demonstrated to be enough for a significant enhancement of fluconazole activity, probably caused by the presence of a low-molecular-weight component in the serum (20
). In this respect, we expanded our present work by adding saliva to the assay medium. We observed only minimal changes in the antifungal activities of the compounds tested, whereas changes in the assay medium or in the pH of the assay medium resulted in considerable variation of antifungal activity (15a
In addition, cooperative effects of lactoferrin with azole agents against Candida
growth were reported by Wakabayashi et al. (41
). Those authors reported on a synergistic activity between lactoferrin and clomitrazole. They demonstrated that 200 μg of lactoferrin per ml alone completely inhibited the growth of Candida
during incubation for 17 h, whereas the addition of clomitrazole (3 to 12 ng/ml) reduced the MIC of lactoferrin to 50 to 100 μg/ml. It was suggested that the interference with proper membrane synthesis by clomitrazole combined with membrane interactions of lactoferrin explains the observed cooperative inhibitory effects of clomitrazole and lactoferrin. In a more recent study the same group also described an increased activity of fluconazole against fluconazole-resistant C. albicans
strains in the presence of lactoferrin (42
). In these experiments the authors observed a synergistic activity against the growth of fluconazole-resistant Candida
strains with 25 to 400 μg of lactoferrin per ml in combination with 0.12 to 0.25 μg of fluconazole per ml after incubation for 15 h in RPMI medium at pH 7.0. These observations compare well with the results of our study with C. albicans
, C. glabratas
, and C. tropicalis
species. On the other hand, in the earlier study this group did not find cooperative effects with the combinations of lactoferrin with amphotericin B or 5-fluorocytosine (41
). Of note, they used only concentrations of up to 100 μg of lactoferrin per ml in their assays to test influences on the MICs of amphotericin B or 5-fluorocytosine. However, in case of amphotericin B, the concentration of 100 μg of lactoferrin per ml might be too low to observe significant changes in MICs (we had to use 500 μg of lactoferrin per ml to observe any effects on the antifungal activity of amphotericin B). In the case of 5-fluorocytosine, the lack of a cooperative effect might be caused by differences in sensitivity of the Candida
strains used to the tested antifungals.
Our study shows that the combination of lactoferrin and amphotericin B can be synergistic up to 30% but may also exhibit a moderate antagonistic activity of 10% against both Candida
species tested. In an earlier study by Nikawa et al. (23
), it was shown that preexposure of C. albicans
to amphotericin B resulted in an increased resistance to apo-lactoferrin-mediated cell death. This observation points to similarities in the antifungal mechanisms of apo-lactoferrin and amphotericin B. It should be realized, however, that the mechanism of Candida
growth inhibition by lactoferrin is not necessarily explained by actions directed at cell surfaces only. In addition, other studies suggested that amphotericin B might also exert its antifungal activity by interference with cellular oxidation (28
). Therefore, differences in the mechanisms of action of lactoferrin and amphotericin B can explain the observed synergistic activity.
Similar reasoning can be put forward for the synergistic activity that we found with the combination of 5-fluorocytosine and lactoferrin. Nikawa et al. (23
) showed that apo-lactoferrin interacts with cell membrane constituents of C. albicans
. Although the details of this mechanism need clarification, the interaction of apo-lactoferrin with the cell membrane might antagonize the effect of antifungals such as 5-fluorocytosine. In our study we observed a pronounced synergistic antifungal effect only with 5-fluorocytosine in combination with lactoferrin. These results might indicate that the antifungal activity of lactoferrin was not likely to be caused by interactions with the cell membrane alone.
Combinations of drugs that show both antagonistic and cooperative activities are difficult to manage in clinical practice, since fluctuations in their levels in plasma and tissue are difficult to control. It should be attractive, therefore, to use lactoferrin in combination with fluconazole, not only because fluconazole is commonly used by patients but also because this combination is likely to exhibit cooperative or even synergistic antifungal activities over the entire range of potential concentrations. During the usual dosage regimens, a mean salivary concentration of 2.6 μg of fluconazole per ml can be reached, which is considerably higher than its MIC against most clinical isolates (9
). In such a situation, the addition of lactoferrin to the existing therapy of fluconazole seems to be of no extra value. However, in case of in vitro resistance of the isolate to fluconazole, resulting in MICs higher than 2.6 μg/ml (Table ) (11
), the addition of lactoferrin can be useful. Our results show that both fluconazole-sensitive and -resistant strains react to the addition of lactoferrin. Therefore, the addition of lactoferrin to the existing therapy with fluconazole may enable a significant lowering of the fluconazole intake and/or may postpone the usual increase in the daily dosage of fluconazole through a delay in induction of resistance against fluconazole.
In an earlier study in our laboratory it was determined that the lactoferrin concentration in saliva was in the range of 10 μg/ml and was not significantly different in healthy and HIV type 1-infected persons. However, it was also demonstrated that larger amounts of Candida
were prevalent in HIV type 1-infected persons with relatively small amounts of lactoferrin in their saliva. On the basis of this observation, it was argued that prophylactic treatment of these patients with additional amounts of lactoferrin might be worthwhile to consider (40
In conclusion, in this study we report on a synergistic activity of fluconazole combined with lactoferrin in vitro against several Candida species. Clinical studies with the aim to elucidate the potential utility of this combination in antimycotic therapy are in progress.