Methylprednisolone Enhances the Growth of Exserohilum rostratum In Vitro, Attenuates Spontaneous Apoptosis, and Increases Mortality Rates in Immunocompetent Drosophila Flies

BRIEF REPORT Methylprednisolone Enhances the Growth of Exserohilum rostratum In Vitro, Attenuates Spontaneous Apoptosis, and Increases Mortality Rates in Immunocompetent Drosophila Flies Dimitrios Farmakiotis,1 Fazal Shirazi,1,a Yanan Zhao,2,a Peguy J. Saad,1 Nathaniel D. Albert,1 Emmanuel Roilides,4 Thomas J. Walsh,3 David S. Perlin,2 and Dimitrios P. Kontoyiannis1 The University of Texas M.D Anderson Cancer Center, Houston; 2Public Health Research Institute Center, New Jersey Medical School, Rutgers–The State University of New Jersey, Newark; 3New York–Presbyterian/Weill Cornell Medical Center, New York City; and 4Aristotle University of Thessaloniki, Greece High concentrations of methylprednisolone (0.32 mg/mL) accelerated growth and attenuated spontaneous apoptosis of Exserohilum rostratum in vitro. Injection of E. rostratum conidia preexposed to 0.32 mg/mL of methylprednisolone for 7 days in immunocompetent flies led to increased mortality and a higher fungal burden. Exposure to methylprednisolone could enhance the virulence of E. rostratum. Keywords. Exserohilum rostratum; virulence; corticosteroids; animal models; apoptosis. Contamination of preservative-free methylprednisolone acetate (MP)–containing vials from a single compounding pharmacy resulted in a multistate outbreak of central nervous system, bone, and joint fungal infections, affecting >700 patients [1–3]. Although a variety of molds were isolated from contaminated vials, the predominant organism was the dematiaceous mold Exserohilum rostratum. Previously, E. rostratum had only been sporadically reported to cause human disease, most commonly in patients with significant immune dysfunction [4, 5]. Data regarding the pathophysiologic mechanisms and, more Received 14 February 2014; accepted 6 May 2014; electronically published 15 May 2014. a F. S. and Y. Z. contributed equally of this report. Correspondence: Dimitrios P. Kontoyiannis, MD, Department of Infectious Disease, Infection Control and Employee Health, Unit 1416, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030 (). The Journal of Infectious Diseases® 2014;210:1471–5 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@ oup.com. DOI: 10.1093/infdis/jiu289 METHODS A clinical isolate of E. rostratum from the cerebrospinal fluid of infected patients ( provided by T. J. W., Weill Cornell Medical Center, New York) was grown on potato agar plates for 5 days at 37°C to obtain conidia. Conidia were collected and washed twice in sterile phosphate-buffered saline (PBS) and resuspended in Roswell Park Memorial Institute (RPMI) medium, with 2% glucose, at a concentration of 103 E. rostratum conidia/mL in the presence of different MP concentrations (0, 0.16, and 0.32 mg/mL). Subsequently, 300 µL were dispensed in 96-well plates and incubated in a plate reader at 37°C for 96 hours; RPMI medium alone was used as the standard control. E. rostratum growth rates were estimated by analyzing absorbance at a wavelength of 600 nm. Spontaneous apoptosis of E. rostratum conidia that were or were not exposed to MP was assessed with DiBAC staining (to determine cell death), dihydrorhodamine 123 (DHR-123) staining (to determine reactive oxygen species [ROS] accumulation), TUNEL staining (to determine DNA fragmentation), and CaspACE FITC-VADFMK staining (to determine metacaspase activity), as previously described (Supplementary Materials) [11]. OregonR wild-type and Toll-pathway-deficient D. melanogaster female flies were used, and standard procedures for their manipulation, feeding, and housing were implemented [9, 10]. We inoculated 2–5-day-old flies (25–30 per group) with E. rostratum conidia, with or without preexposure to high-dose MP (0.32 mg/mL) in liquid PBS for 7 days at room temperature, by injecting their lateral thoracic wall with a 10-µm needle that had been dipped in a solution containing 106 conidia/mL (Supplementary Figure 1). BRIEF REPORT • JID 2014:210 (1 November) • 1471 1 specifically, the effect of corticosteroids on the growth and lethality of this unusual pathogen are scarce [4]. In previous public health emergencies, data from in vitro experiments and animal models have been useful in providing the scientific basis for effective management of life-threatening infections [6]. As fungi have been postulated to express steroid receptors that could affect their growth and/or virulence in the presence of corticosteroids [4, 7, 8], we investigated the effect of MP on in vitro growth, spontaneous apoptosis, and in vivo lethality of E. rostratum. To that end, we inoculated E. rostratum conidia, with or without 7-day exposure to MP, into Drosophila melanogaster flies, a minihost model that has been previously shown to simulate the pathogenesis of a variety of invasive mold infections [9, 10]. rates were compared by means of 2-way analysis of variance. PCR-based conidial equivalent counts were compared with the Mann–Whitney test. Kaplan–Meier survival curves for flies were compared with the log–rank test. For all comparisons, 2-tailed P values of <.05 were considered statistically significant. RESULTS Compared with controls (which were not exposed to MP), the growth of E. rostratum was significantly enhanced in the presence of a high (0.32 mg/mL) but not low (0.16 mg/mL) concentration of MP (P = .0046; Figure 1A). MP attenuated spontaneous apoptosis of conidia, as evidenced by the significantly lower proportion of apoptotic dead cells (determined by DiBAC staining; Figure 2A and 2E ), ROS accumulation (determined by DHR-123 staining; Figure 2B and 2E ), DNA fragmentation (determined by TUNEL staining; Figure 2C and 2E ), and decreased metacaspase activity (determined by CaspACE Figure 1. Methylprednisolone (MP) enhances the in vitro growth of Exserohilum rostratum and increases the mortality rate in an immunocompetent fly model of disseminated infection: A, Growth curves of E. rostratum in the absence of corticosteroids or in the presence of 2 different doses of MP. B, Survival curves of wild-type (WT) Drosophila flies injected in their lateral thoracic wall with 106 E. rostratum conidia/mL (P < .001 by the log–rank test). C, Representative hematoxylin-eosin (HE) and Gomori methenamine silver (GMS) stains of WT fly abdominal sections, showing a significantly higher fungal burden (arrows) in a fly infected with conidia of E. rostratum preexposed to 0.32 mg/mL of MP in phosphate-buffered saline (PBS) for 7 days (bottom, MP), compared with a fly infected with conidia of E. rostratum incubated for 7 days in PBS alone (top, control). D, Results of quantitative polymerase chain reaction analysis of whole flies injected with E. rostratum preexposed to 0.32 mg/mL of MP for 7 days in PBS (E. rostratum + MP) or PBS alone (control). Data are conidia equivalents/500 µL of fly homogenate, and horizontal lines den (...truncated)