Antifungal properties of selective serotonin reuptake inhibitors against Aspergillus species in vitro

C. Lass-Flörla,*, M. P. Diericha, D. Fuchsb, E. Semenitzc, I. Jeneweina and M. Ledochowskid

a Departments of Hygiene and Social Medicine and b Medical Chemistry and Biochemistry, University of Innsbruck, Innsbruck; Departments of c Hospital Pharmacy and d Clinical Nutrition, Innsbruck University Hospital, Innsbruck, Austria


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study investigated the fungicidal activity of selective serotonin reuptake inhibitors (SSRIs) against clinical isolates of Aspergillus fumigatus (n = 11), Aspergillus flavus (n = 9), Aspergillus terreus (n = 10) and Candida parapsilosis (ATCC 22019). The common drugs fluoxetine, seroxate, sertraline, paroxetine and reboxetine were applied in a broth microdilution test. In addition, we examined whether a post-antibiotic effect occurs following short exposure to the drugs. The various SSRIs showed time- and dose-dependent effects and were fungicidal towards the organisms tested. Sertraline and fluoxetine were the most active drugs, yet there were differences in the susceptibility of the various isolates tested. A lag of regrowth was dependent on the various SSRIs tested and their concentration. Treatment for 4 h at concentrations of sertraline below and equipotent to the minimal fungicidal concentration resulted in a lag of regrowth of 8–24 h for isolates of A. fumigatus and A. flavus. In conclusion, our in vitro studies clearly demonstrate antifungal effects of SSRIs. Animal studies are needed to evaluate the potential role of these psychotropic drugs in the management of fungal infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Years ago antimicrobial activity was described for psychotropic drugs of the phenothiazine and thioxanthene groups.1 Since then, several non-antibacterial substances have been examined, and it was reported that selective serotonin reuptake inhibitors (SSRIs) influence the in vitro viability of bacteria2–4 and may reverse chloroquine resistance in Plasmodium falciparum.5 These drugs have a significant antimicrobial activity, mainly against Gram-positive bacteria, yet are inactive against most enteric Gram-negative bacteria.4

SSRIs are increasingly being used as first-line therapy for severe premenstrual syndrome (PMS) and as antidepressants.6 Recently, we found that sertraline (SSRI, Tresleen, Vienna, Austria) has in vivo and in vitro antifungal activity against Candida spp.7 In this study we examined whether SSRIs also exert fungicidal effects against Aspergillus spp. conidia and hyphae. The common drugs fluoxetine, seroxate, paroxetine, sertraline and reboxetine were investigated. In addition, we studied whether a post-antibiotic effect results following short exposure to the various drugs tested.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains

The in vitro tests were carried out on clinical isolates of Aspergillus fumigatus (n = 10), Aspergillus flavus (n = 9), Aspergillus terreus (n = 10) and Candida parapsilosis (ATCC 22019). Isolates were maintained as suspensions in water at room temperature, and subcultures were grown on Sabouraud glucose agar (Merck, Darmstadt, Germany) and incubated at 35°C for 5 days. One isolate of A. fumigatus (isolate AF 72) resistant to itraconazole in vitro and in vivo, kindly provided by D. W. Denning, was included in the study.

Drugs

Sertraline HCl (mol. wt 343 kDa), fluoxetine HCl (mol. wt 346 kDa), paroxetine HCl (mol. wt 365 kDa), citaloprame HCl (mol. wt 324 kDa) and reboxetine methansulphonate (mol. wt 410 kDa) were kindly provided by their respective manufacturers. The drugs were dissolved in sterile aqua ad injectionem (Fresenius, Linz, Austria) at room temperature for 30 min and centrifuged. Depending on the solubility of the drug substances (manufacturers' information) stock solutions were obtained and serial two-fold drug dilutions of the supernatant were prepared: sertraline HCl, 1900–7 mg/L; fluoxetine HCl and citaloprame HCl, 5000– 19 mg/L; paroxetine HCl, 4000–15 mg/L; and reboxetine methansulphonate, 1000–3 mg/L.

Broth microdilution tests for conidia

Fungi were tested in a modified broth microdilution method as described previously.8,9 The conidial suspension was harvested by flooding each colony with 2 mL of sterile 0.85% saline. Turbidity was measured with a spectrophotometer at 350 nm (DU-64 Spectrophotometer; Beckman, Toulerton, MN, USA); transmission was adjusted with sterile 0.85% saline to 78–82% for A. flavus, 87–82% for A. fumigatus and A. terreus, and 78–72% for C. parapsilosis. The suspension was further diluted in RPMI 1640 medium to obtain 1.2–8 x 105 cfu/mL for Aspergillus spp. and 3.2–4 x 104 cfu/mL for C. parapsilosis. A total of 100 mL of each of the drug dilutions was inoculated with 100 mL of the fungal suspensions, and the mixture was incubated at 35°C. Final concentrations used were 950–3 mg/L for sertraline, 2500–9 mg/L for fluoxetine and citaloprame, 2000–7 mg/L for paroxetine and 500–1.5 mg/L for reboxetine.

To determine the minimal fungicidal concentration (MFC), 100 µL volumes were taken from every well at 8, 24 and 48 h of incubation and spread on Sabouraud glucose agar. The number of cfu was counted after incubating the plates at 35°C for 72 h until growth of subcultures from the growth control well was apparent. The MFC was defined as the lowest drug concentration at which 99% of the inoculum was killed. Each experiment was done twice and carried out in duplicate.

Broth microdilution test for hyphae

The conidial stock solutions were prepared as described above in RPMI 1640 containing 10 mM HEPES (Sigma, St Louis, MO, USA). A modified culture technique was used as described earlier.9 Briefly, 100 µL of conidial suspensions were added to 96-well plates (Costar, Vienna, Austria), and the plates were incubated at 30°C for 16–22 h for the formation of hyphae. At this time, drug concentrations were added similarly to conidial tests and the MFC was determined as described above. Each experiment was done twice and carried out in duplicate.

Post-antibiotic effect

Conidial suspensions were prepared as described above and incubated with the various antidepressants for 1 and 4 h at 35°C. Lag of regrowth was assessed using a modification of the procedure of Nagl et al.10 Concentrations equipotent to, one dilution above and one below the MFC for each isolate were investigated. Afterwards, fungi were washed twice with sterile water, centrifuged at 4000g for 2 min and refilled with RPMI 1640. Quantitative cultures of non-diluted samples and 1:100 and 1:1000 dilutions in aqua were spread on Sabouraud glucose agar, incubated at 35°C and examined visually for growth every 12 h. We compared the time required for colony count of untreated and treated isolates and examined the cultures for a lag of regrowth. Each experiment was done twice and carried out in duplicate.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Broth microdilution tests for conidia and hyphae

The SSRIs showed time- and dose-dependent effects and were fungicidal towards the tested fungi, as shown in Table 1Go. Sertraline, followed by fluoxetine, were the most active drugs with isolate-dependent in vitro susceptibility. For sertraline the MFC ranges at 8 h (48 h) for Aspergillus conidia and hyphae were 14–475 mg/L (3–237 mg/L) and 7–118 mg/L (3–59 mg/L), respectively. For sertraline the MFC for C. parapsilosis was 29–59 mg/L during 48 h of incubation. Paroxetine, citaloprame and reboxetine showed comparable MFCs, yet with higher values. The MFC ranges of the various SSRIs at 24 h were similar to the MFCs at 48 h for Aspergillus conidia and hyphae (data not shown).


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Table 1. In vitro activity of the selective SSRIs against 30 isolates of Aspergillus spp.
 
Post-antibiotic effect

Lag of regrowth depended on the individual SSRIs and the concentration tested, as shown in Table 2Go. Treatment for 4 h with sertraline at concentrations below and equipotent to the MFCs (48 h) for conidia showed a lag of regrowth of 8–24 h for some isolates. No effects were seen after an exposure time of 1 h. Concentrations higher than the MFC resulted in a lag of regrowth and/or a decrease in cfu count for sertraline. A lag of regrowth for fluoxetine, reboxetine, paroxetine and citaloprame was shown for some isolates after an exposure time of 1 and 4 h.


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Table 2. Post-antibiotic effects of Aspergillus spp. (n = 30) treated with different concentrations of the various SSRIs for 1 and 4 h
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The five SSRIs tested in this study displayed different potencies with respect to both antifungal killing and lag of fungal regrowth. Sertraline and fluoxetine showed the highest activity against Aspergillus spp. and C. parapsilosis with differences in susceptibility of the various isolates tested.

A number of non-antibiotic drugs such as anti-inflammatory drugs,11 mucolytic agents12 and proton pump inhibitors13 exert an influence on the physiology and viability of bacteria. The precise mechanism by which these drugs affect bacteria is not yet known,14 and activity against fungi has not been reported before to our knowledge.

In humans, SSRIs modify the behaviour of 5-hydroxytryptamine (5HT) in the synapse space.15 These antidepressants act primarily at the 5HT transporter protein (SERT) and block the reuptake process of 5HT.14,15 SERTs, with mol. wts of 60–80 kDa and 12 transmembrane domains are similar to other biogenic amine transporters and part of sodium- and chloride-dependent transporters.15 It is probable that antifungal activity results from an interaction of SSRIs and fungal transporter systems, as has been noted for Staphylococcus aureus and chlorpromazine.14

We observed, however, that the various neuronal monoamine reuptake inhibitors were active against the two types of inocula tested. Recently, we found that in order to kill hyphae in vitro, antifungals had to be applied at significantly higher concentrations.9 Surprisingly, similar phenomena were not seen for the various SSRIs tested. In contrast, MFCs for conidial inocula were higher as compared with hyphal inocula in some isolates, as shown in Table 1Go. Importantly, our data show that the broad antifungal effect is also exerted on the itraconazole-resistant A. fumigatus strain.

Considering the bioavailability of these antidepressant drugs, it remains unclear whether our in vitro findings are of relevance in vivo. The maximal achievable concentrations range between 0.1 mg/L for fluoxetine and 0.05 mg/L for sertraline,15 and were clearly lower than the MFCs for the strains tested. Nevertheless, concentrations of sertraline in the cerebrospinal fluid and brain are >40-fold higher (2 mg/L) as compared with plasma levels.16 It is possible that these serum levels may be sufficient to modify fungal virulence. A lag of regrowth was observed after exposure to the agents, and the extent of this effect depended on the concentration and incubation time of the various psychotropic substances. The maximum duration of lag of regrowth was observed at incubation times shorter than those required for killing, but at concentrations similar to the MFC.

In conclusion, SSRIs act against Aspergillus spp. in at least two steps: reversible attenuation and, if incubation is prolonged, irreversible changes resulting in loss of viability. Our in vitro findings probably provide a rationale for the local treatment of fungal infections with formulae containing SSRIs. Animal models and clinical trials are highly warranted to evaluate the potential role of SSRIs in the management of fungal infections. Identification of the mode of action could be of great help in the development and research of new antifungal drugs.


    Notes
 
* Corresponding author. Tel: +43-512-507-3425; Fax: +43-512-507-2870; E-mail: Cornelia.Lass-Floerl{at}uibk.ac.at Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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7 . Lass-Flörl, C., Dierich, M., Fuchs, D., Semenitz, E. & Ledochowski, M. (2001). Antifungal activity against Candida species of the selective serotonin reuptake inhibitor sertraline. Clinical Infectious Diseases, in press.

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9 . Lass-Flörl, C., Nagl, M., Speth, C., Ulmer, H., Dierich, M. P. & Würzner, R. (2001). Studies of in vitro activities of voriconazole and itraconazole against Aspergillus hyphae using viability staining. Antimicrobial Agents and Chemotherapy 45, 124–8.[Abstract/Free Full Text]

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Received 8 May 2001; returned 5 July 2001; revised 14 August 2001; accepted 30 August 2001