Potentiated inhibition of Trichoderma virens and other environmental fungi by new biocide combinations

Abstract

Fungi cause diverse, serious socio-economic problems, including biodeterioration of valuable products and materials that spawns a biocides industry worth ~$11 billion globally. To help combat environmental fungi that commonly colonise material products, this study tested the hypothesis that combination of an approved fungicide with diverse agents approved by the FDA (Food and Drug Administration) could reveal potent combinatorial activities with promise for fungicidal applications. The strategy to use approved compounds lowers potential development risks for any effective combinations. A high-throughput assay of 1280 FDA-approved compounds was conducted to find those that potentiate the effect of iodopropynyl-butyl-carbamate (IPBC) on the growth of Trichoderma virens; IPBC is one of the two most widely used Biocidal Products Regulations-approved fungicides. From this library, 34 compounds in combination with IPBC strongly inhibited fungal growth. Low-cost compounds that gave the most effective growth inhibition were tested against other environmental fungi that are standard biomarkers for resistance of synthetic materials to fungal colonisation. Trifluoperazine (TFZ) in combination with IPBC enhanced growth inhibition of three of the five test fungi. The antifungal hexetidine (HEX) potentiated IPBC action against two of the test organisms. Testable hypotheses on the mechanisms of these combinatorial actions are discussed. Neither IPBC + TFZ nor IPBC + HEX exhibited a combinatorial effect against mammalian cells. These combinations retained strong fungal growth inhibition properties after incorporation to a polymer matrix (alginate) with potential for fungicide delivery. The study reveals the potential of such approved compounds for novel combinatorial applications in the control of fungal environmental opportunists. KEY POINTS: • Search with an approved fungicide to find new fungicidal synergies in drug libraries. • New combinations inhibit growth of key environmental fungi on different matrices. • The approach enables a more rapid response to demand for new biocides.

Keywords: Aspergillus brasiliensis; Aureobasidium pullulans; Biodegradation; Biodeterioration; Chaetomium globosum; Fungicide; Penicillium funiculosum.

Fig. 1

High-throughput chemical screening for agents that potentiate fungal growth inhibition by IPBC. (A) The scatterplots show the normalised growth yield (OD₆₀₀) of T. virens for each PL compound in the absence (x axis) and presence (y axis) of IPBC, at 40 h and 72 h. The data are means from duplicate screens. (B) Effect strengths of the 1280 combinations; those that showed an effect strength >50 at 40 h are coloured in pink, at 72 h in blue (in A and B) and at both time points in orange (panel on the right ). Agents selected for further tests are indicated: TZ, thioridazine; FZ, flunarizine; TFZ, trifluoperazine; PZ, prochlorperazine; HEX, hexetidine. The full dataset is in Supplemental Table S1

Fig. 2

Library compound chemical interactions with IPBC in several environmental fungi. The colour gradient relates to the combinatorial effect strength for each combination, determined after 40 h growth of the fungus indicated. Combinatorial effect strength was calculated as described in the “Methods”. Data are means from at least three independent experiments. TZ, thioridazine, FZ, flunarizine, TFZ, trifluoperazine, PZ, prochlorperazine, HEX, hexetidine

Fig. 3

Trifluoperazine and hexetidine do not potentiate the effect of IPBC in a human cell line. Cells were incubated for 24 h in the presence of the compounds alone or in combination. Cell viability was then measured using a CCK-8 kit. The values are means ± SEM from three replicate experiments. The combinatorial effect strength for both combinations is <12. There were no significant differences between the conditions according to multiple comparisons (Tukey’s test) by one-way ANOVA

Fig. 4

Halofantrine and quinine interactions with IPBC against the ASTM G21 fungi. After 40 h growth of the five indicated fungi, combinatorial effect strength was calculated as described in the “Methods” for the combinations halofantrine + IPBC and quinine + IPBC. The values are means ± SEM from at least three independent experiments

Fig. 5

The activity of the combinations IPBC + HEX/TFZ is retained after coating on glass with sodium alginate. Round glass covers were coated with 0.25% (w/v) sodium alginate supplemented with 250 μg ml⁻¹ IPBC, 15 μM HEX, and/or 15 μM TFZ alone or in combination. Spores of T. virens were then sprayed on to surfaces of the coated covers. Images were taken after 14 days. The four replicate images for each condition are from independent experiments