Medical Mycology, 2017, 0, 1–8 doi: 10.1093/mmy/myx057 Advance Access Publication Date: 0 2017 Original Article Original Article In vivo pathogenicity of Trichosporon asahii isolates with different in vitro enzymatic profiles in an immunocompetent murine model of systemic trichosporonosis Alexandra M. Montoya1 , Carolina E. Luna-Rodrı́guez1 , Rogelio de J. Treviño-Rangel1 , Miguel Becerril-Garcı́a1 , Raquel G. Ballesteros-Elizondo2 , Odila Saucedo-Cárdenas2 and Gloria M. González1,∗ 1 Departamento de Microbiologı́a and 2 Departamento de Histologı́a, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, México ∗ To whom correspondence should be addressed. Gloria M. González, PhD, Av. Madero Pte. s/n esq. Dr. Aguirre Pequeño, Col. Mitras Centro, 64460 Monterrey, Nuevo León, México. Tel/Fax: +52 (81) 8329-4177 / (81) 8676-8605; E-mail: firstname.lastname@example.org Received 5 December 2016; Revised 24 March 2017; Accepted 13 July 2017; Editorial Decision 12 April 2017 Abstract Trichosporon asahii is an opportunistic yeastlike fungus that colonizes the gastrointestinal and respiratory tracts and human skin. Although it is an important cause of disseminated infections by non-Candida species, there are a few reports related to its virulence factors and their possible role in in vivo pathogenicity. We developed a murine model of disseminated trichosporonosis in immunocompetent mice for the evaluation of the in vivo pathogenicity of 6 T. asahii isolates with different in vitro virulence factor profiles. Tissue fungal burden was determined on days 1, 3, 7, 15, and 25 post-challenge. Overall, the largest fungal load was detected in the kidney on the 5 experimental days, while brain, spleen, and liver displayed a comparatively low fungal count. We observed a fungal burden decrease in most experimental groups from day 15. Histological analysis showed the presence of T. asahii in tissue and a generalized inflammatory infiltrate of polymorphonuclear cells in the kidney, liver, red pulp of the spleen, and the hippocampus. Even though our isolates showed different in vitro virulence factors profiles, we did not detect relevant differences when assayed in vivo, except for a higher persistence of a proteaseand biofilm-producing strain in kidney, liver, and brain. Key words: Trichosporon asahii, murine model, trichosporonosis, tissue fungal burden, enzymatic profiles, biofilm. Introduction Trichosporon asahii is an opportunistic yeastlike fungus that colonizes the gastrointestinal and respiratory tracts, and human skin. It is commonly associated with white piedra and systemic infections in immunocompromised patients,1 although fungemia in immunocompetent patients has been reported.2,3 Systemic infections by Trichosporon C The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: email@example.com 1 2 Medical Mycology, 2017, Vol. 00, No. 00 Table 1. Enzymatic profiles of the six Trichosporon asahii isolates included in this study. Pz valuea Strain GenBank accession no. Isolation site Biofilm producer DNAse Protease Esterase Hemolysin Phospholipase G-1995 07-230 09-206 09-928 09-1238 12-1238 KM269302 KM269305 KM269313 KM269317 KM269318 KM269331 Urine Skin Urine Urine Urine Urine Weak Strong Very strong Strong Weak Intermediate Positive Positive Positive Positive Positive Positive Strong Negative Negative Very strong Negative Negative Very strong Very strong Very strong Very strong Very strong Very strong Negative Very strong Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Pz value: ≤0.69 = very strong, 0.70–0.79 = strong, 0.80–0.89 = mild, 0.90–0.99 = weak, 1.00 = negative. spp. have a mortality of 50–80%4 and are considered the second or third most common yeast-associated fungemias in immunocompromised patients, particularly those with hematological diseases.5–8 Neutropenia is the main risk factor; however, an important association has been established between trichosporonosis and the use of invasive devices such as catheters and probes, and chemotherapy or corticosteroid treatment.4,9,10 Other factors that may predispose to the development of trichosporonosis include solid tumors, transplants, peritoneal dialysis, and human immunodeficiency virus (HIV) infection.11 Despite T. asahii being considered an important cause of disseminated infections by non-Candida species, there are a few reports related to its pathogenicity, and most involve immunosuppressed mice.12–14 These studies report a variable tissue dissemination and fungal burden in relation to the different inocula used. Gokaslan and Anaissie determined that a ≥2 × 107 cfu inoculum had a 100% mortality rate and organ invasion in immunosuppressed mice on day 6 post-infection.15 On the other hand, Hospenthal et al. reported that a ≥7 × 106 cfu inoculum was needed to reach a 100% mortality rate and tissue dissemination at 6 h postinfection.14 Yamagata et al. observed that an inoculum of 3 × 106 cfu sufficed to reach a 80% mortality rate in immunosupressed mice.13 These models were developed using different mice strains, immunosuppression schemes, inocula, and T. beigelii, an obsolete taxon for T. asahii. All these factors may account for the differences in infectivity and disease evolution observed in the various trichosporonosis murine models. Nevertheless, there are no comparative studies on the pathogenicity of different T. asahii strains that may determine whether variability among members of this species exists. In the present study, our goal was to develop a murine model of disseminated trichosporonosis in immunocompetent mice for the in vivo evaluation of the variable pathogenicity of T. asahii clinical isolates. We aimed to establish a model that allowed the survival of the mice and systemic dissemination of the microorganism; could monitor the course of infection based on quantification of fungal burden in organs; and could compare histological analysis for the determination of pathogenicity differences among the T. asahii strains evaluated. Methods Clinical isolates Six pathogenic strains of Trichosporon asahii were included in this study: G-1995, 07–230, 09–206, 09–928, 09–1238, and 12–1238. All are pathogenic isolates from catheter related urinary infections, except for strain 07–230, which was isolated from a skin infection. The strains were chosen based on differential phenotypic characteristics such as lytic enzymes16 and biofilm production [A. M. Montoya, unpublished results]. Phenotypic characteristics, clinical source, and GenBank accession numbers for each strain are listed in Table 1. The isolates had been previously identified by morphologic features, carbohydrate assimilation profiles, and intergenic spacer 1 (IGS1) sequence analysis. The strains were stored in sterile water at room temperature. Enzymatic determinations The in vitro evaluation of extracellular lytic compounds was performed as in a previous study.16 DNAse was assessed with DNAse test agar with methyl green medium (Becton Dickinson, Le Pont de Claix, France); hemolytic activity was assayed using Sabouraud blood medium;17 phospholipase activity was carried out on egg yolk medium;18 aspartyl-protease activity was evaluated using bovine serum albumin medium;19 and esterase activity was evaluated on Tween 80 medium.20 Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 90028 were included as positive controls, and sterile deionized water was used as a negative control for all enzymatic tests. All assays were conducted twice in independent tests using fresh cultures of each strain. Each independent test included technical duplicates for every sample. Activity was classified according to a semi-quantitative Pz value, which is the ratio of the colony diameter to the Montoya et al. diameter of the activity halo. Activity was thus categorized as very strong (Pz ≤ 0.69), strong (Pz = 0.70–0.79), mild (Pz = 0.80–0.89), weak (Pz = 0.90–0.99), and negative (Pz = 1.0). Biofilm production Biofilm production was performed with modifications to the method by DiBonaventura.21 T. asahii strains were subcultured in Sabouraud glucose agar (SGA) at 30◦ C for 24 h, transferred to yeast extract broth (YEPD; 1% yeast extract, 2% peptone, 2% glucose), and incubated overnight at 30◦ C and 150 rpm. Cells were collected by centrifugation and washed with sterile phosphate buffered saline (PBS) before being resuspended in RPMI 1640 with L-glutamine buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (RPMI 1640-MOPS; Hardy Diagnostics, Santa Maria, California, USA). The inoculum was adjusted to 1 × 107 cfu/ml by spectrophotometry, and 100 μl were later transferred to sterile 96-well polystyrene flat bottom plates (Corning Incorporated, Corning, New York, USA). Plates were incubated for 4 h at 37◦ C, after which the media was decanted and wells were washed twice with 150 μl of PBS to remove nonadhered cells. One hundred μl of RPMI 1640-MOPS were added to each well and plates were incubated at 37◦ C for 72 h, substituting the RPMI 1640-MOPS every 24 h. After 72 h of incubation, plates were washed twice with PBS and left to dry for 1 h at 37◦ C. Quantification of biofilm was carried out as described by Melo et al.22 Wells were dyed with 110 μl of an aqueous solution of 0.4% crystal violet (CV), and left at room temperature for 45 min. The CV solution was decanted, and the wells were washed thrice with 200 μl of sterile distilled water. To dissolve the dye, 200 μl of 95% ethanol were added to each well and these were incubated at room temperature for 45 min. One hundred μl of this solution was transferred to a clean plate and quantified by spectrophotometry at 595 nm. Experiments were performed twice in independent tests using fresh cultures of each strain. Each independent test included technical duplicates for every sample. C. albicans ATCC 90028 was used as an internal control. Biofilm production was arbitrarily categorized based on the absorbance readings. Thus, biofilm phenotypes were classified as weak (A595nm ≤ 0.531), intermediate (A595nm = 0.532–0.763), strong (A595nm = 0.764–0.999), and very strong (A595nm ≥ 1.0) 3 approximately 18–20 g. Mice were distributed five per cage in 12 h light/dark cycles and provided with sterilized food and water ad libitum. Care, maintenance, and handling of the animals were in accordance with the Mexican government’s license conditions for animal experimentation and the Guide for the Care and Use of Laboratory Animals. Experiments were authorized by the Ethics and Research Committee, Facultad de Medicina, Universidad Autónoma de Nuevo León (registration number: MB13-004). The six Trichosporon asahii isolates were subcultured from water stocks onto potato dextrose agar (PDA) plates and incubated at 30◦ C for 48 h to determine the culture’s purity and viability. This growth was then transferred to YEPD and incubated at 37◦ C and 200 rpm for 24 h. The resulting cells were obtained by centrifugation at 3500 rpm for 10 min, washed two times with PBS, and resuspended in 0.85% NaCl. The number of conidia was counted with a hemocytometer and diluted to a final inoculum of 5 × 106 cfu/ml, with ≥95% of conidia (arthroconidia and blastoconidia). Cell counts were verified by serial dilution in PDA plates and incubated at 30◦ C for 24 h. Aliquots of 0.2 ml of adjusted inoculum (1 × 106 cfu/mice) of each of the strains were inoculated into the lateral tail veins of six groups of 25 mice. Ten control animals were injected with sterile saline. On days 1, 3, 7, 15, and 25 postinfection, five mice per experimental group and two control animals were selected at random and killed by cervical dislocation. The spleens, livers, brains, and kidneys of each of these animals were removed aseptically. Both spleens and median lobes of livers were cut into two symmetric parts; hemispheres of the brains were divided by a sagittal cut; and both kidneys of each mouse were cut transversally into their upper and lower halves. Tissue fungal burden analysis One half of the liver, spleen, and brain, and the upper half of the left kidney were immediately weighed after removal, transferred to 2 ml of 0.85% NaCl, mechanically homogenized (Polytron-Aggregate, Kinematica), and serially diluted. Aliquots of the undiluted and diluted homogenates were plated onto PDA plates and incubated at 30◦ C for 48 h. The colonies were counted, and the number of cfu per gram of each organ was calculated. Histopathology Systemic model of trichosporonosis We used 160 4-week-old BALB/c male mice (purchased from Envigo, Somerset, New Jersey, USA) weighing One half of the removed liver, spleen, and brain, and the lower half of the left kidneys were immediately fixed with 4% buffered formalin. Samples were dehydrated, 4 paraffin embedded, and sliced into 5 μm sections, which were then stained with hematoxylin-eosin and GrocottGomori’s methenamine silver stain. Histological analysis was restricted to the observation of evidence of tissue damage, inflammatory infiltrates, and the presence of fungal structures in tissue. Statistical analysis Tissue fungal burden counts of the evaluated organs in the different experimental groups were analyzed statistically using the Kruskal-Wallis test and the Dunn’s multiple comparisons test. Correlation between fungal burden and putative virulence factors was evaluated using the Spearman test. All statistical analyses were performed in GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, California, USA). A P-value < .05 was considered significant. Results Strains used in this study were chosen on the basis of their in vitro virulence profiles shown in Table 1. All strains were positive for DNAse and showed no phospholipase activity. Strain 07–230 had very strong hemolysin activity; strain 09–206 displayed the highest esterase, and strain 09–923 showed very strong aspartyl-protease activity. Regarding their biofilm producing phenotype, both strains G-1995 and 09–1238 showed weak biofilm production; strain 12–1238 had an intermediate biofilm producing capacity, and strains 07–230, 09–206, and 09–928 were either strong or very strong biofilm producers. A 7-day pilot study was carried out to determine the inocula that would be used for our experimental model. BALB/c mice were infected with the following cfu/mouse doses of the randomly chosen T. asahii 07–230 strain: 5 × 105 , 7 × 105 , 1 × 106 , 5 × 106 , 1 × 107 , and 5 × 107 . Mice inoculated with the lowest dose did not always become fungemic, which was tested for by plating blood on PDA plates. Furthermore, this inoculum had a complete clearance of fungi in all organs from all mice at 7 dpi. In contrast, the three highest doses tested (5 × 106 to 5 × 107 ) had mortality rates of ≥40% by day 2 post-infection. Thus, the 1 × 106 inoculum was selected for the trichosporonosis murine model on the basis of being the higher dose that (1) allowed for the survival of all infected mice, (2) showed fungal persistence on 7 dpi, and (3) was capable of mounting a systemic infection as evidenced by fungal burden in blood. The pathogenicity of six T. asahii strains with different in vitro virulence profiles was evaluated in the murine model of trichosporonosis. Mice were monitored daily during the course of the experimental model. No animals died in the Medical Mycology, 2017, Vol. 00, No. 00 25-day period. Pilo-erection was noticeable on mice during the first 4 dpi, and no weight loss or obvious neurological impairments were observed. Results for tissue fungal burden are shown in Figure 1. Overall, the highest fungal load of all strains was detected in the kidney, followed by the brain, liver, and spleen on the 25 experimental days. Strains showed a significant decrease in fungal count by day 15 when compared to 1 dpi (P < .05), except for kidney. Strains 07–230, 09–928, and 12–1238 showed a significant decrease of fungal burden in kidney until 25 dpi (P < .05). Kidney showed no fungal clearance at 25 dpi for any of the strains evaluated. Fungal clearance in spleen was complete at 25 dpi for most mice, as we observed persistence of T. asahii in 40% of mice infected with strain 12–1238. At the same time point, incomplete fungal clearance was observed in the liver in 40% and 80% of mice infected with strains 12– 1238 and 09–928, respectively. Similarly, 60% of mice inoculated with strain 07–230 showed fungal persistence in the brain. Strain 09–928 showed the highest cfu counts for kidney, spleen, and liver on day 1 post-infection. This strain also maintained a higher fungal persistence in kidney, liver, and brain at day 25 when compared to other strains. No correlation could be established between fungal burden and hemolysin or biofilm production (P ≥ .2). Aspartylprotease production correlated to fungal burden in the liver and brain on 25 dpi (P < .0001). Although no significant association could be established between protease production and fungal burden in kidney, the protease-producing strain 09–928 had the highest fungal load in this organ on day 25. Histological analysis by hematoxylin-eosin and GrocottGomori’s methenamine silver stain are depicted in Figures 2 and 3. Staining with hematoxylin-eosin showed inflammatory infiltrate characterized by polymorphonuclear cells in renal corpuscles and proximal tubes of the cortex in kidney; in splenic sinusoids and splenic cords of the red pulp in spleen; and in cords of hepatocytes and sinusoids in the liver. Analysis of the brain showed no anomalies in the cortex, striatum, or cerebellum. Inflammatory infiltrate was observed only in between neurons and around blood vessels of the hippocampus. No structural anomalies were observed in any of the organs. Regarding the presence of T. asahii in tissue, analysis showed scarce conidia and hyphal fragments in kidney and liver on the sites of inflammatory infiltrate. Hyphal fragments were also observed in the red pulp and surrounding the lymph nodes of the white pulp in the spleen. In the brain, hyphal fragments were present in the cortex and cerebellum. Notably, we could not observe fungal structures in the hippocampus. Montoya et al. 5 Figure 1. Means of fungal burden in the kidneys, spleens, livers and brains from BALB/c mice intravenously infected with 1 × 106 cfu/mouse of the strains determined on day 1, 3, 7, 15, and 25 post-challenge. Discussion Trichosporon sp. is mainly associated to white piedra; however, in recent decades T. asahii has acquired relevance as a causative agent of systemic opportunistic infections since the first trichosporonosis case reported in 1970.23 Its broad geographical distribution, resistance to some antifungals, and increase of risk groups are factors that have made the study of species within this genus relevant. Herein we aimed to determine if a difference in pathogenicity within members of the species exists. To this, we developed a murine model of disseminated trichosporonosis in immunocompetent mice using isolates with different in vitro virulence profiles. Experimental models of disseminated trichosporonosis have been developed principally in mice, and most of these focus on the therapeutic efficacy of antifungal compounds24–27 or on the pathogenicity of immunosuppressed animals.12–14 Gokaslan and Anaissie included immunocompetent CF1 mice in their murine model of disseminated trichosporonosis.15 Although they used a strain identified with the old 6 Medical Mycology, 2017, Vol. 00, No. 00 Figure 2. Representative histopathological hematoxylin-eosin stained sections of the kidneys, spleens, livers and brains from BALB/c mice intravenously infected with 1 × 106 cfu/mouse of T. asahii 09–928. Scale bar equals 50 μm. H: hippocampus. This Figure is reproduced in color in the online version of Medical Mycology. Figure 3. Representative histopathological Grocott-Gomori’s methanamine silver stained sections of the kidneys, spleens, livers and brains from BALB/c mice intravenously infected with 1 × 106 cfu/mouse of T. asahii 09–928. Scale bar equals 50 μm. S: striatum; C: cortex. This Figure is reproduced in color in the online version of Medical Mycology. Montoya et al. nomenclature of Trichosporon beigelii and outbred mice, their findings on its pathogenicity are similar to ours. A dose of 1 × 106 cfu/mouse had a 100% survival rate at day 30, with a higher fungal count in kidney compared to liver and spleen; however, they found a complete clearance in these two organs by day 7, whereas we observed fungal persistence in the liver and spleen throughout the course of the experimental model in some mice infected with strains 09–928 and 12–1238, albeit with low cfu counts. They also reported a lowered fungal count in kidney by day 6 postinfection followed by an increase at day 8. In contrast, all our fungal counts including kidney showed a tendency to decrease over time. Overall, our results suggest that infections by T. asahii in immunocompetent mice were resolved around 7 dpi. One of the most prevalent signs in invasive trichosporonosis has been granulomatous inflammation in the liver and spleen of both immunocompromised and immunosuppressed mice, as well as fungal structures collocated with the lumen or periphery of blood vessels.14,15,28 Histological analysis allowed the observation of scarce inflammatory infiltrate of polymorphonuclear cells in all organs evaluated. The presence of these cells was associated with the location of fungal structures in both kidney and liver. In our model, fungal structures in relation to blood vessels could only be observed in the brain. The scarcity of fungal structures also relates to the low cfu counts for all organs on the five experimental days evaluated. Mariné et al. developed a murine model comparing three different T. asahii strains in immunocompromised mice but found no difference in their virulence based on their fungal burden and pathological findings. Their study, however, does not state why these three strains were selected, or whether there was a phenotypical difference among them.29 Similarly, our results did not reveal differences amongst isolates for tissue damage, inflammatory infiltrate, nor fungal presence. Although no differences on histological findings could be observed amongst our isolates, we do report persistence of fungal burden for strain 09–928 in the kidney, liver and brain, albeit with low counts. Strain 09–928 had the distinction of being a strong or very strong producer of aspartylprotease and biofilm, unlike the other strains evaluated. The virulence role of aspartyl-proteases has been extensively described for C. albicans, where it has been demonstrated that they participate in host protein degradation, adherence to different cell types and tissues, and the regulation of phenotype switching.30 On the other hand, biofilm production relates to a facilitated colonization, growth and proliferation of microorganisms, as well as increased resistance against antifungals and environmental stress.31 Biofilm production has also been associated to a high mortality rate in systemic 7 infections by C. albicans.32,33 The infective capacity of several fungi hinges on diverse mechanisms that may help during nutrient consumption, tissue invasion, adherence, resistance, and dissemination in the host.34 The study of these virulence factors has contributed to the understanding of the pathogenesis during infections of C. albicans,35 Cryptococcus spp.,36 and Aspergillus fumigatus,37 to name a few. There are no reports of in vivo production by T. asahii for any lytic enzyme or biofilm. Whether or not aspartylproteases and biofilms have a role in the pathogenicity in vivo of T. asahii cannot be concluded from this study, and should be further explored. Herein, we compare the pathogenicity of six T. asahii strains differing in their in vitro virulence profile. Although the strains showed differences in their hemolytic, protease, and biofilm production in vitro, we did not detect relevant differences when assayed in vivo, except for a higher persistence of a protease- and biofilm-producing strain in kidney, liver and brain. Further studies are needed to validate a possible association between protease and biofilm production and higher virulence. Acknowledgment We thank Sergio Lozano-Rodriguez, MD, and Alejandro QuirogaGarza, MD, of the “Dr. José E. González” University Hospital (Monterrey, México) for their review of the manuscript prior to submission. Funding This work was supported by the Consejo Nacional de Ciencia y Tecnologı́a [CONACyT-INFRA-2015-01-251142]. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. 2. 3. 4. 5. Walsh TJ, Groll A, Hiemenz J et al. Infections due to emerging and uncommon medically important fungal pathogens. Clin Microbiol Infec. 2004; 10 (Suppl. 1): 48–66. Wolf DG, Falk R, Hacham M et al. Multidrug-resistant Trichosporon asahii infection of nongranulocytopenic patients in three intensive care units. J Clin Microbiol. 2001; 39: 4420–4425. Rastogi VL, Nirwan PS. Invasive trichosporonosis due to Trichosporon asahii in a non-immunocompromised host: a rare case report. Indian J Med Microbiol. 2007; 25: 59–61. Ruan S-Y, Chien J-Y, Hsueh P-R. Invasive trichosporonosis caused by Trichosporon asahii and other unusual Trichosporon species at a medical center in Taiwan. Clin Infect Dis. 2009; 49: e11–17. 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