Synergistic antifungal activity between posaconazole and caspofungin or FK506 against <i>C. albicans</i> drug susceptible or resistant strains.

#<p>Orange highlighting color indicates wild-type and its derived mutants, while yellow and green highlighting colors represent echinocandin resistant and 3 (#1, #9, #17) out of 17 azole resistant isolates from the strain series, respectively.</p>*<p>Formula of FIC index:</p><p>[(MIC₁₀₀ of drug A in combination)/(MIC₁₀₀ of drug A alone)]+[(MIC₁₀₀ of drug B in combination)/(MIC₁₀₀ of drug B alone)].</p><p>FIC≤0.5 (Synergy); FIC >4 (Antagonism); FIC >0.5 but ≤4 (no interaction).</p

<i>Candida albicans</i> strains used in this study.

<p><i>Candida albicans</i> strains used in this study.</p

Minimum and fractional inhibitory concentrations of compounds against <i>Streptomyces scabies</i> PS07.

<p>Minimum and fractional inhibitory concentrations of compounds against <i>Streptomyces scabies</i> PS07.</p

<i>B</i>. <i>amyloliquefaciens</i> Ba01 showed antibacterial activity against <i>S</i>. <i>scabies</i> causing potato common scab.

<p>A. Phylogenetic trees based on either the (a) 16S rRNA sequence or (b) <i>gyrA</i> gene sequence showed the evolutionary relationships between <i>Bacillus</i> isolates. The numbers at the nodes represent bootstrap values. The scale bars indicating the numbers of substitutions per nucleotide position were (a) 0.005 and (b) 0.02. <b>B.</b> Multiple <i>Bacillus</i> isolates exhibited a diverse degree of antibacterial activity against <i>S</i>. <i>scabies</i> PS07. Disk diffusion assays were used to test the antibacterial activity of <i>Bacillus</i> species against <i>S</i>. <i>scabies</i>. In this experiment, 10⁶ <i>S</i>. <i>scabies</i> spores in 100 μL were spread on solid YME medium, and 3 μL of 1 OD₆₀₀ (~6x10⁴ cells) of <i>Bacillus</i> isolates were loaded on the right disk, while 3 μL of ddH₂O were loaded on the left disk as a control. <b>C.</b> Ba01 was selected to test its antibacterial activity against multiple <i>S</i>. <i>scabies</i> isolates. <i>S</i>. <i>scabies</i> isolates were spread on solid YME medium, and 3 μL of 1 OD₆₀₀ of Ba01 were added to the right disk and ddH₂O to the left disk. All plates were incubated at 28°C for five days and photographed.</p

<i>B</i>. <i>amyloliquefaciens</i> Ba01 inhibited the growth and sporulation of <i>S</i>. <i>scabies</i> PS07.

<p><b>A.</b> A disk diffusion assay was used to observe the antibacterial effects of Ba01 against <i>S</i>. <i>scabies</i> PS07 on solid YME medium. Clear (C) and undifferentiating (U) zones were observed around the disk loaded with the Ba01 isolate. <b>B.</b> The morphology of Ba01 from panel A was observed under a scanning electron microscope. <b>C.</b> <i>S</i>. <i>scabies</i> PS07 without Ba01 treatment (ddH₂O treated) produced spiral hyphae and sporulation septa with constrictions. <b>D.</b> <i>S</i>. <i>scabies</i> PS07 in the undifferentiating zone exhibited vegetative/smooth hyphae and septa without constrictions. Resolution is 10,000X. The scale bar represents 1 μm.</p

Strains used in this study.

<p>Strains used in this study.</p

Surfactin, iturin A, and fengycin inhibited the growth and formation of spiral hyphae of <i>S</i>. <i>scabies</i> PS07.

<p><b>A.</b> Disk diffusion assays were used to test the anti-<i>S</i>. <i>scabies</i> activity of surfactin, iturin A, and fengycin. Approximately 10⁶ <i>S</i>. <i>scabies</i> spores were spread on YME solid agar, and a 6-mm disk containing iturin A (dissolved in ethanol), surfactin (dissolved in methanol), or fengycin (dissolved in methanol) were pressed on the surface of an agar plate and incubated at 28°C for five days. <b>B.</b> The morphology of <i>S</i>. <i>scabies</i> PS07 in the undifferentiating zone of panel A was observed under a scanning electron microscope. (a) The magnification is 5,000X, and the scale bar represents 10 μm. (b) The magnification is 15,000X, and the scale bar represents 2.5 μm.</p

Ba01 reduced the disease severity of potato common scab in pot assays.

<p>The growth conditions of <i>S</i>. <i>scabies</i> PS07, Ba01, and potato plants were described in the materials and methods. <b>A.</b> Three five-week-old potato plants were used for each treatment: (a) mock control without inoculation of <i>S</i>. <i>scabies</i> PS07 or Ba01; (b) inoculation of <i>S</i>. <i>scabies</i> PS07 only; (c) inoculation of Ba01 only; (d) inoculation of PS07 and Ba01 on the same day; and (e) inoculation of PS07 first, and then Ba01 inoculation after 14 days. The bar represents 1 cm. <b>B.</b> The disease severity and disease incidence of the potato common scab were reduced when potato plants were inoculated with PS07 and Ba01 on the same day. Data were expressed as the average of tubers collected from three potato plants ± standard error of the mean. <i>P</i> values were calculated using Tukey's test. Asterisks (**) indicate <i>P</i> < 0.01 as compared to the treatment of PS07 only.</p

Calcineurin is required for drug tolerance in <i>C. lusitaniae</i>.

#<p>Cells were grown overnight at 30°C and washed twice with dH₂O. Then 0.5 OD (in 500 µl) of cells was spread on RPMI 1640 media (Remel; R04067). After 20 min, the E-test strips (bioMérieux Corp.) were transferred to the surface of the media. The minimum inhibitory concentrations (MIC) were read after 24 h incubation at 35°C according to the manufacturer’s instructions.</p

Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in <em>Candida lusitaniae</em>

<div><p><em>Candida lusitaniae</em> is an emerging fungal pathogen that infects immunocompromised patients including HIV/AIDS, cancer, and neonatal pediatric patients. Though less prevalent than other <em>Candida</em> species, <em>C. lusitaniae</em> is unique in its ability to develop resistance to amphotericin B. We investigated the role of the calcium-activated protein phosphatase calcineurin in several virulence attributes of <em>C. lusitaniae</em> including pseudohyphal growth, serum survival, and growth at 37°C. We found that calcineurin and Crz1, a <em>C. albicans</em> Crz1 homolog acting as a downstream target of calcineurin, are required for <em>C. lusitaniae</em> pseudohyphal growth, a process for which the underlying mechanism remains largely unknown in <em>C. lusitaniae</em> but hyphal growth is fundamental to <em>C. albicans</em> virulence. We demonstrate that calcineurin is required for cell wall integrity, ER stress response, optimal growth in serum, virulence in a murine systemic infection model, and antifungal drug tolerance in <em>C. lusitaniae</em>. To further examine the potential of targeting the calcineurin signaling cascade for antifungal drug development, we examined the activity of a calcineurin inhibitor FK506 in combination with caspofungin against echinocandin resistant <em>C. lusitaniae</em> clinical isolates. Broth microdilution and drug disk diffusion assays demonstrate that FK506 has synergistic fungicidal activity with caspofungin against echinocandin resistant isolates. Our findings reveal that pseudohyphal growth is controlled by the calcineurin signaling cascade, and highlight the potential use of calcineurin inhibitors and caspofungin for emerging drug-resistant <em>C. lusitaniae</em> infections.</p> </div