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JAC Advance Access originally published online on September 19, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):1058-1061; doi:10.1093/jac/dkl384
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp.

Christina Kratzer1, Selma Tobudic1, Monika Schmoll2, Wolfgang Graninger1 and Apostolos Georgopoulos1,*

1 Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy, Medical University of Vienna Währinger Gürtel 18-20, 1090 Vienna, Austria 2 Institute of Chemical Engineering, Vienna University of Technology Getreidemarkt 9, 1060 Vienna, Austria

*Corresponding author. Tel: +43-1-40400-5139; Fax: +43-1-40400-5200; E-mail: apostolos.georgopoulos{at}meduniwien.ac.at

Received 21 April 2006; returned 26 May 2006; revised 7 June 2006; accepted 30 August 2006


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Objectives: The uncommon fungal pathogen Trichoderma shows increasing medical importance particularly in immunocompromised patients. Despite systemic antifungal therapy, prognosis of Trichoderma infection is poor regardless of the type of infection and the therapy used. The aim of the present study was to evaluate the in vitro activity and synergism of double antifungal combinations including amphotericin B, voriconazole, fluconazole, chlorhexidine digluconate and Akacid plus® against 15 isolates of Trichoderma longibrachiatum and 1 isolate of Trichoderma harzianum.

Methods: Individual MICs were determined by using broth microdilution method following the NCCLS M38-A guidelines with standard RPMI 1640 broth. Synergy tests were performed using the chequerboard method.

Results: All clinical Trichoderma strains showed reduced susceptibility to fluconazole (MICs ≥ 64 mg/L) and amphotericin B (MICs = 2 mg/L), whereas lower MICs of 0.5–1 mg/L were detected for voriconazole. Akacid plus® reached the lowest MIC values in a range of 0.06–0.5 mg/L, 4- to 32-fold higher MICs were found for chlorhexidine. No antagonism was observed for any of the antifungal combinations tested. Interaction of amphotericin B and azoles was indifferent (fractional inhibitory concentration index, FICI 2–4). The combination of one azole and one cationic biocide showed different degree of synergism (FICI 0.07–2.03). Interaction of Akacid plus® and chlorhexidine resulted in synergism for each Trichoderma isolate (FICI-range 0.05–0.5).

Conclusions: These results demonstrate no interaction between antifungals and some degree of synergism between azoles and cationic antimicrobials against Trichoderma spp.

Keywords: filamentous fungi , MICs , voriconazole , chlorhexidine , Akacid plus®


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Trichoderma species are saprophytic filamentous fungi with worldwide distribution in the soil, plant material, decaying vegetation and wood. Although Trichoderma is commonly considered as a contaminant, several case studies on infections in humans with this uncommon mould have revealed that this fungal pathogen shows increasing medical importance particularly in immunocompromised patients, such as neutropenic cases and transplant recipients as well as patients with chronic renal failure.1 The genus Trichoderma has five medically relevant species Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma pseudokoningii and Trichoderma viride. T. longibrachiatum is the main human pathogen species within the genus.

The treatment of Trichoderma infections in humans requires systemic antifungal therapy, removal of the foreign bodies, surgical intervention and therapy of the underlying disease.2–5 Despite antimicrobial therapy with amphotericin B alone or in combination, prognosis of Trichoderma infection is poor regardless of the type of infection and the therapy used.1 Previous reports on antifungal susceptibility testing against T. longibrachiatum have documented elevated MICs of conventional antimycotics such as amphotericin B, fluconazole, itraconazole and flucytosine.2,5 Also, various biocides including the cationic antiseptics show fungicidal activity against mould pathogens.6 Although combination antifungal therapy is used in some cases of systemic Trichoderma infections, in vitro synergy tests have not been performed so far.

The aim of the present study was to investigate the in vitro activity and synergism of double antifungal combinations including conventional antimycotics such as amphotericin B, voriconazole, fluconazole and cationic antimicrobials such as the bisbiguanide chlorhexidine digluconate and the novel polymeric guanidine Akacid plus® against 15 isolates of T. longibrachiatum and 1 isolate of T. harzianum.


   Materials and Methods
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Fungi

Twelve clinical isolates of T. longibrachiatum including ATCC 201044 and ATCC 208859, one clinical isolate of T. harzianum (CBS 102174) and three environmental isolates of T. longibrachiatum CECT 2937, CECT 20105 and CECT 2606 were tested. Clinical or environmental origin of these isolates is shown in Table 1. The identity of all strains of T. longibrachiatum and T. harzianum was confirmed by internal transcribed spacer sequence (GenBank accession numbers AY328034 [GenBank] ,-35,-37,-38,-39,-40,-41,-42; AY585879 [GenBank] ,-80; AY920396 [GenBank] ,-97,-98; Z82912 [GenBank] ; X93929 [GenBank] ) analysis. T. longibrachiatum IP 2110.92 and CNM-CM-382, which were originally identified as T. pseudokoningii4 and T. koningii,7 were reidentified as T. longibrachiatum based on ITS sequence analysis and PCR-fingerprinting.


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  		Table 1. Individual MICs of amphotericin B (AMB), voriconazole (VRC), fluconazole (FLC), chlorhexidine digluconate (CHG) and Akacid plus® (AP) against 13 clinical and 3 environmental Trichoderma isolates

 
Antifungal susceptibility testing

Amphotericin B (Bristol-Myers Squibb, Epernon, France), fluconazole (Pfizer, Amboise, France) and voriconazole (Pfizer) were purchased as standard powders. Stock solutions of amphotericin B and voriconazole were prepared in 100% dimethyl sulphoxide, and fluconazole powder was dissolved in distilled water. Akacid plus®, a 3:1 mixture of poly-(hexamethylen-guanidinium-chloride) and poly-[2-(2-ethoxy)-ethoxyethyl)-guanidinium-chloride], (PoC, Vienna, Austria) and chlorhexidine digluconate (Sigma, St Louis, MO, USA) were acquired as 25% and 20% aqueous solutions.

The individual MICs were determined initially by using broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) for moulds with standard RPMI 1640 broth buffered to pH 7 with 0.165 M MOPS and supplemented to 2% glucose.8 Inoculum conidial stock suspensions were prepared in sterile saline containing 1% polysorbate 80 from fresh, mature (5- to 7-day-old) cultures on potato dextrose agar at 25°C. The inoculum size was adjusted to an optical density that ranged from 0.09 to 0.13 (0.5–2.5 x 106 non-germinated conidia/mL suspension). The suspensions were then diluted 1:50 to obtain a final working inoculum of 1–5 x 104 cfu/mL. Fungal inocula (100 µL) were added to each well of the microdilution tray, and each well contained 100 µL of drug solution (2x final concentration). The plates were incubated at 35°C and were read visually after 48 h of incubation. MICs were defined as the lowest concentration of the antifungal agent that completely inhibited the fungal growth. Drug-free and fungus-free controls were included; quality control was ensured by testing Aspergillus niger ATCC 16404, A. niger ATCC 10578, A. fumigatus ATCC 14110, Candida krusei ATCC 6258 and Candida tropicalis ATCC 750, respectively.

Chequerboard tests were employed to determine the in vitro efficacy of each double combination for each test isolate. Quantitative analysis of antifungal combinations was performed as described previously.9 The fractional inhibitory concentration (FIC) of each drug for an individual isolate was calculated as the ratio of the MIC of the drug in combination to the MIC of the drug alone. The FIC index (FICI) value for an individual isolate was calculated by adding the FICs of both tested antifungals. FICI values were interpreted as follows: FICI ≤ 0.5, synergistic; 0.5 < FICI ≤ 4, indifferent and FICI > 4 antagonistic.

All susceptibility tests were performed in triplicate; results were accepted only when there was not more than a one-step difference in values. If this was the case, the higher value was reported.


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Antimicrobial activity

Results of the individual MICs of conventional and cationic antimicrobials are illustrated in Table 1. Fluconazole reached the highest MIC values ranging from 64 to 256 mg/L against all tested isolates of T. longibrachiatum, even a higher MIC of 1024 mg/L was found against T. harzianum CBS 102174. Apart from only one environmental strain, all clinical Trichoderma isolates showed reduced susceptibility to amphotericin B (MICs = 2 mg/L). Lower MICs of 0.5–1 mg/L were detected for voriconazole. The novel biocide Akacid plus® reached MIC values ranging from 0.06 to 0.5 mg/L, whereas 4- to 32-fold higher MICs were determined for chlorhexidine digluconate.

Overall synergy results of Trichoderma isolates are summarized in Table 2. No antagonism was observed for any of the antifungal combinations tested. In vitro interaction of amphotericin and azoles was indifferent for all Trichoderma strains; FICI values ranging from 2 to 4 were determined. Voriconazole and fluconazole in combination also appeared to be indifferent for most strains (FICI50 = 2). The synergy tests of one conventional antifungal and one cationic antimicrobial resulted in a synergistic or indifferent effect. Interactions of azoles and chlorhexidine were synergistic for most strains (FICI50 = 0.28 or 0.31) and synergistic to indifferent for all strains (FICI90 = 0.54 or 0.88). In general, azoles MICs in combination decreased one to nine 2-fold dilutions, whereas chlorhexidine in combination decreased only slightly (one to three 2-fold dilutions). Interactions of azoles with Akacid plus® were synergistic in 25% and 37.5% of the tested isolates. The combination of both cationic antimicrobials showed synergism for all Trichoderma isolates (FICI90 = 0.39). Akacid plus® MICs in combination decreased three to seven 2-fold dilutions, while chlorhexidine MICs decreased only two to five 2-fold dilutions.


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  		Table 2. FICI results of double combinations for Trichoderma test isolates (n = 16)

 

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The present study demonstrated reduced activity of fluconazole and amphotericin B and higher in vitro activity of voriconazole against clinical and environmental Trichoderma isolates. Tested MIC values of these conventional antifungals were comparable to those previously reported in the literature for this species.2,5 According to the CLSI document, no breakpoints exist for the definition of resistance in filamentous fungi.8 The difficulty in treating invasive mould infections could be explained because of poor penetration of antifungal agents into infected tissue. Voriconazole which is registered for the treatment of systemic Aspergillus, Candida, Fusarium spp. and Scedosporium infections penetrates well in the central nervous system. Due to lower MIC values, fewer side effects and higher penetration than amphotericin B, voriconazole might be an important drug in the treatment of invasive Trichoderma infections.

Previous in vitro studies on combination of antifungal drugs against the most common mould species Aspergillus spp. have resulted in controversial findings.9 Our synergy tests revealed indifferent interaction between amphotericin B and azoles against Trichoderma spp. The combination of voriconazole or fluconazole with Akacid plus® or chlorhexidine showed different degrees of synergism, whereas the interaction of both cationic antimicrobials was synergistic for each Trichoderma isolate.

Although some cases of superficial or localized Trichoderma infections such as sinusitis,3 stomatitis, infection of the skin2 and otitis externa1 are reported in literature, mostly the diagnosis of Trichoderma spp. is not made until dissemination of the fungi where fine septate hyphae are found in lungs, brain, heart, liver, stomach and pretracheal abscesses. An earlier diagnosis of Trichoderma spp. could enable the clinical application of cationic antimicrobials to support the treatment of localized Trichoderma infections in the future.


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None to declare.


   Acknowledgements
 
We thank Waltraud Schmidt at the Clinical Department for Infectious Diseases and Chemotherapy for excellent technical advice.


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1 Chouaki T, Lavarde V, Lachaud L, et al. (2002) Invasive infections due to Trichoderma species: report of 2 cases, findings of in vitro susceptibility testing, and review of the literature. Clin Infect Dis 35:1360–7.[CrossRef][Web of Science][Medline]

2 Munoz FM, Demmle GJ, Travis WR, et al. (1997) Trichoderma longibrachiatum infection in a pediatric patient with aplastic anemia. J Clin Microbiol 35:499–503.[Abstract/Free Full Text]

3 Furukawa H, Kusne S, Sutton DA, et al. (1998) Acute invasive sinusitis due to Trichoderma longibrachiatum in a liver and small bowel transplant recipient. Clin Infect Dis 26:487–9.[Web of Science][Medline]

4 Gautheret A, Dromer F, Bouris JH, et al. (1995) Trichoderma pseudokoningii as a cause of fatal infection in a bone marrow transplant recipient. Clin Infect Dis 20:1063–4.[Web of Science][Medline]

5 Richter S, Cormican MG, Pfaller MA, et al. (1999) Fatal disseminated Trichoderma longibrachiatum infection in an adult bone marrow transplant patient: species identification and review of literature. J Clin Microbiol 37:1154–60.[Abstract/Free Full Text]

6 Kratzer C, Tobudic S, Graninger W, et al. (2006) In vitro antimicrobial activity of the novel polymeric guanidine Akacid plus. J Hosp Infect 63:316–22.[CrossRef][Web of Science][Medline]

7 Campos-Herrero MI, Bordes A, Perera A, et al. (1996) Trichoderma koningii peritonitis in a patient undergoing peritoneal dialysis. Clin Microbiol Newslett 18:150–1.[CrossRef]

8 National Committee for Clinical Laboratory Standards. (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-forming Filamentous Fungi: Proposed Standard M38-A(NCCLS, Wayne, PA, USA).

9 Johnson MD, MacDougall C, Ostrosky-Zeichner L, et al. (2004) Combination antifungal therapy. Antimicrob Agents Chemother 48:693–715.[Free Full Text]

10 Guarro J, Antolin-Ayala MI, Gene J, et al. (1999) Fatal case of Trichoderma harzianum infection in a renal transplant recipient. J Clin Microbiol 37:3751–5.[Abstract/Free Full Text]


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