Comparative pathogenicity of Scedosporium species in murine model of systemic scedosporiosis

Abstract

Systemic scedosporiosis is a devastating emerging fungal infection caused by several species of the genus Scedosporium in immunocompetent and immunocompromised individuals. In this study, we compared the virulence of different Scedosporium species in a murine model of systemic scedosporiosis by survival assays, fungal burden and histopathological analysis. We found that mice mortality was species-dependent, S. apiospermum, S. aurantiacum and S. dehoogii were the most virulent species. We also observed the dissemination and invasion of Scedosporium species to the brain, spleen and kidney by colony count and histopathological analysis at different times of infection. Particularly, the brain was the tissue most susceptible to invasion during systemic scedosporiosis. This study shows the virulence and pathophysiology of different Scedosporium species and will be useful in facilitating control and prevention strategies for systemic scedosporiosis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01065-4.

Introduction

Scedosporium species are filamentous fungi that have been found in high human-active areas worldwide and are considered relevant emerging opportunistic fungal pathogens causing severe infections in immunocompetent and immunosuppressed individuals, known as scedosporiosis [1–3]. Recently, the World Health Organization considers Scedosporium spp a priority pathogen, and several species increasingly recognized as the second most opportunistic mold fungus after Aspergillus species [4]. The genus Scedosporium currently consists of several species: S. apiospermum, S. aurantiacum, S. boydii, S. cereisporum, S. dehoogii, S. angustum, S. desertorum, S. ellipsoideum, S. fusoideum, S. minutisporum, S. rarisporum, and S. sanyaens, among others species without clinical significance [5, 6]. Scedosporiosis cause a wide variety of clinical conditions, from cutaneous and soft tissue infection to systemic and disseminated infections [7, 8]. The most severe clinical manifestation of scedosporiosis is the disseminated infection, which may also involve the central nervous system [9]. Disseminated scedosporiosis is difficult to treat and frequently fatal, there has been an increase in the reporting of new cases worldwide in latest years. Moreover, a high degree of antifungal resistance in systemic scedosporiosis has increasingly been reported over the past decade [2, 5, 8]. The species of Scedosporium causing infections have been undergoing constant epidemiological changes over the years, and also studies have suggested that the clinical presentation, severity, risk factors and outcome of mycosis is dependent on the species of Scedosporium [10, 11]. Therefore, there is an imperative clinical need to understand the virulence and pathogenesis in different Scedosporium species in order to have a better understanding of the mycosis.

Since Scedosporium spp have emerged as a cause of serious human infections, numerous in vivo virulence studies have been performed in models such as invertebrates as vertebrate animals. Those studies evidenced that Scedosporium species differ in virulence and pathogenicity [12–14]. In this study, we evaluated the pathogenicity of five Scedosporium species in a mouse model of systemic infection. We found that mortality of infected animals, fungal burden and fungal invasion to tissues were fungal species-specific, and we showed that the brain was the tissue most susceptible to dissemination and invasion by Scedosporium species.

Materials and methods

Strains

A total of ten strains of Scedosporium species were included in this study: two strains of S. apiospermum (00320, 120MS), S. aurantiacum (175HG, 118DF), S. dehoogii (103NL, 003NL) S. boydii (081370, 112CL) and S. angustum (147OC, 169JC). All strains were identified and confirmed by sequencing the ITS, D1/D2 and β-tubulin gene region, previously published data [15]. For the preparation of the inoculum, the strains were incubated on potato dextrose agar for seven days at 30 °C. Conidial cells were harvested with sterile saline and the suspension was filtered with sterile gauze and centrifuged. The conidia were counted with a hemocytometer and adjusted to the final inoculum concentration required. To corroborate the conidial counts, serial dilutions were cultured on potato dextrose agar plates at 30 °C.

Mice

Five week-old ICR male mice (weighing 20–22 g; purchased from Envigo, Mexico) were used for the in vivo studies. Mice were housed 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: MB11-008).

Systemic model of scedosporiosis

Groups of ten mice were inoculated intravenously with 200 µL of 1 × 10⁶ conidia of each of the strains of Scedosporium and five mice were injected with sterile saline (control group) via the lateral tail vein. After injection, each experimental group were returned to the cages and monitored daily for survival evaluation. Deaths were registered until day 30 post-infection. Animals that survived after day 30 post-infection were sacrificed.

Fungal burden assay

Groups of twelve mice were injected intravenously with 200 µL of 5 × 10⁴ conidia of each of the strains of Scedosporium and nine mice injected with sterile saline (control group) via the lateral tail vein. After infection, groups of four mice infected with each of strains of Scedopsorium and three mice of control group were sacrificed on day 3, 6 and 15 post-infection. Fungal burden determination in kidney, spleen and brain. All organs were aseptically removed, half of each organ was weighed and mechanically homogenized (Polytron-aggregate, Kinematica) in 500 µL sterile saline. Homogenates were used to prepare 1:10 serial dilutions, 50 µL of each dilution were plated by duplicate on potato dextrose agar. Colony-forming units (CFUs) were determined after 7 days of incubation at 30 °C and results were expressed as log10 cfu/g tissue. No colonies were recovered from tissues of uninfected mice control group. All experiments were performed independently at least twice.

Histopathology

Half of each organ obtained from infected or uninfected mice (above referred), were immediately removed and fixed with 4% buffered formalin. Samples were dehydrated, paraffin-embedded and sliced into 5-μm sections. The sections were stained with Grocott methenamine silver (Merck Millipore, Darmstadt, Germany) for light microscopy observations following the manufacturer’s specifications.

Statistics

Mean survival time (MST) was estimated by the Kaplan–Meier method and compared among groups by the log-rank test. The fungal tissue burdens of the tested organs in the different experimental groups were analyzed using the Kruskal–Wallis test in SPSS (version 17.0 for Windows; Chicago, IL, USA). P ≤ 0.05 was statistically significant.

Results

Adult mice were injected intravenously with different Scedosporium species to compare in vivo their pathogenicity. We observed 100% of death of mice infected with two isolates of S. apiospermum, the strain of S. apiospermum 00320 was more lethal than 120MS causing death at 5 day post-infection (dpi) (Fig. 1A and Supplementary 1), similarly, 100% of mice inoculated with two strains of S. aurantiacum (118DF and 175HG) died at 3 and 13 dpi, respectively (Fig. 1B and Supplementary 1). Meanwhile, we found 100% of death of mice injected with S. dehoogii 103NL at 4 dpi compared to 80% of death of isolate S. dehoogii 003NL at 24 dpi (Fig. 1C and Supplementary 1). In contrast, 90% of mice infected with both strains of S. boydii (112CL and 081370) died at 13 and 18 dpi, respectively (Fig. 1D and Supplementary 1). In group of mice injected with S. angustum, we showed 90% of death of mice infected with strain 147OC at 13 dpi compared to 60% of death of isolate 169JC at 24 dpi (Fig. 1E and Supplementary 1). S. apiospermum, S. aurantiacum and S. dehoogii were the most virulent species causing 100% death of mice during the first 5 days of infection, compared to S. boydii and S. angustum which were less virulent (Fig. 1F and Supplementary 1).

Fig. 1.

Survival of mice with systemic scedosporiosis. Survival of mice infected intravenously with 1 × 10⁶ cfu of A S. apiospermum, B S. aurantiacum, C S. dehoogii, D S. boydii and E S. angustum. F Survival of animals infected with the most virulent strains of each Scedsoporium species tested. n = 10 mice per experimental group

In order to evaluate the dissemination of the fungus to tissues during systemic infection, we decided to choose the most virulent strain of each Scedosporium species and prepared fungal burden assay. The results are summarized in Table 1. Briefly, we showed that brain was the tissue with the highest fungal burden during infection, followed by the spleen and kidney of mice infected with each of Scedosporium species. The brain of mice infected with S. apiospermum 00320 and S. aurantiacum 118DF evidenced an increase in fungal burden at 6 and 15 dpi, in contrast to the brain of mice inoculated with S. dehoogii 103NL and S. angustum 147OC there was no difference during infection. On the other hand, the kidney of mice infected with S. apiospermum 00320 showed an increase in fungal burden at 15 dpi, meanwhile, the spleen showed no differences in CFUs during infection, similarly, the spleen and kidney of mice infected with S. aurantiacum 118DF no differences in CFUs were observed. Finally, in the spleen and kidney of mice infected with S. dehoogii 103NL, S. boydii 112CL and S. angustum 147OC we found a decrease in fungal burden at days 6 and 15 dpi.

Table 1.

Fungal tissue burden in mice intravenously infected with 5 × 10⁴ conidia of Scedosporium species

Strain (GenBank accession number)	Tissue	Log CFU/g tissue (median range)
		3 dpi	6 dpi	15 dpi
S. apiospermum 00320 (KX620428)	Brain	4.88 (4.80 – 5.38)	5.30 (4.93 – 5.72)	6.26 (5.93 – 6.43)
	Spleen	3.73 (3.25 – 4.18)	3.45 (3.22 – 3.56)	3.61 (2.90 – 4.32)
	Kidney	2.92 (2.56 – 3.20)	3.72 (3.61 – 3.73)	4.05 (3.55 – 4.80)
S. aurantiacum 118DF (KX620383)	Brain	3.99 (3.57 – 4.08)	4.95 (4.60 – 5.18)	5.27 (4.62 – 5.73)
	Spleen	3.40 (3.28 – 3.63)	3.17 (3.11 – 3.40)	3.20 (3.11 – 3.64)
	Kidney	2.77 (2.50 – 2.84)	2.69 (2.49 – 2.87)	2.51 (2.45 – 2.58)
S. dehoogii 103NL (KX620377)	Brain	3.90 (3.74 – 4.04)	3.84 (3.61 – 4.14)	3.82 (3.60 – 4.34)
	Spleen	4.00 (3.87 – 4.08)	3.88 (3.44 – 3.98)	3.66 (3.50 – 4.18)
	Kidney	3.28 (2.95 – 3.62)	3.04 (2.90 – 3.30)	3.10 (3.04 – 3.18)
S. boydii 112CL (KX620385)	Brain	4.15 (3.68 – 4.32)	4.48 (4.14 – 4.61)	2.68 (2.58 – 2.85)
	Spleen	3.94 (3.63 – 4.04)	2.73 (2.34 – 3.11)	2.52 (2.32 – 3.18)
	Kidney	2.97 (2.66 – 3.30)	2.90 (2.72 – 3.15)	2.98 (2.81 – 3.36)
S. angustum 147OC (KX620370)	Brain	4.17 (3.70 – 4.40)	3.68 (3.61 – 3.74)	3.59 (3.50 – 3.60)
	Spleen	4.43 (4.32 – 4.57)	4.16 (4.14 – 4.45)	4.02 (3.70 – 4.08)
	Kidney	3.60 (3.30 – 3.74)	3.54 (2.62 – 3.56)	3.29 (3.15 – 3.53)

CFU Colony forming-unit

dpi day post-infection

To demonstrate the tissue invasion of Scedosporium species, we performed a histopathological analysis with Grocott's methenamine-silver staining to reveal fungal structures. We observed fungal structures, mainly hyphae in spleen and kidney of mice infected with each of the Scedosporium species at 3 dpi, whereas by days 6 and 15 post-infection the presence of fungal structures was scarce (data not shown). Interestingly, the brain was the tissue with the highest invasion of fungal structures during infection in mice infected with each of Scedosporium species (Fig. 2). The brain of mice infected with S. apiospermum 00320 showed abundant presence of hyphae invading the tissue at 3, 6 and 15 dpi. Meanwhile, the brain of mice infected with S. aurantiacum 118DF and S. dehoogii 103NL evidenced moderate presence of hyphae in the parenchyma during infection. Compared to the brain of mice infected with S. boydii 112CL and S. angustum 147OC scarce fungal structure were observed during infection (Fig. 2). These results showed that S. apiospermum, S. aurantiacum and S. dehoogii are the most virulent species of the Scedosporium complex, which caused 100% of the death of the animals during the first days of infection, and also we showed that the most susceptible tissue to Scedosporium invasion was the brain by fungal burden and histological assays. S. apiospermum was the strain that presented high levels of fungal burden and tissue invasion in the brain parenchyma during infection.

Fig. 2.

Histopathology of the brain during murine systemic scedosporiosis. Histopathological brain sections stained with Grocott methenamine silver–stain from uninfected or infected ICR mice intravenously with 5 × 10⁴ conidia of the most virulent strains of each Scedsoporium species tested at 3, 6 and 15 days post-infection, showing hyphal elements

Discussion

Systemic scedosporiosis is a potentially devastating emerging mycosis that usually affects immunocompromised patients, although it has also been affected in immunocompetent individua near-drowning victims. An increase in cases of Scedosporium infections has been reported over the last two decades, and it has been shown that the clinical presentation, severity and outcome of mycosis is dependent on the Scedosporium species [10]. In this study, we evaluated the virulence of different Scedosporium species in a murine model of systemic infection by survival assay, fungal burden and histopathological assay in different tissues during infection.

Previous murine models of systemic scedosporiosis have shown that S. apiospermum, S. aurantiacum and S. dehoogii were the most virulent species and caused death in immunocompetent animals during the first six days of infection, whereas S. boydii was the least virulent species [16]. As seen in this study, S. apiospermum, S. aurantiacum and S. dehoogii were the most virulent species, while S. boydii and S. angustum were the less virulent. Several models of systemic scedosporiosis have demonstrated the dissemination of the fungus to different tissues such as kidney, spleen and brain [17, 18]. In this study by fungal burden assay, we evidenced that all five Scedoporium species were found in spleen, kidney and brain during days 3, 6 and 15 post-infection, being the brain the tissue with the highest fungal burden during infection. Particularly, S. apiospermum and S. aurantiacum were the species that originated the highest levels of fungal burden in the brain compared to other species. Nweze et al. in their murine model of systemic infection with S. apiospermum observed high mortality rates, and also demonstrated the ability of the S. apiospermum to disseminate to the brain of mice [17]. The ability of different Scedoporium species to invade the human brain of patients with disseminated scedosporiosis has been previously demonstrated [19–21], showing marked neurotropism and a high propensity to cause central nervous system infections.

Cerebral scedosporiosis is one of the most frequent clinical manifestations of systemic S. apiospermum and S. aurantiacum infections, which is difficult to resolve and usually fatal [22]. Central nervous system infections usually manifest as multicentric or solitary brain abscesses. Rodriguez et al. in their murine model of systemic Scedosporiosis using three clinically relevant species such as S. boydii, S. aurantiacum and S. apiospermum evidenced that the brain was the most affected tissue and the histopathological analysis showed the presence of hyphae in blood vessels and brain parenchyma, a higher fungal invasion was observed in the tissue of mice infected with S. aurantiacum [18]. In this study, we showed scarce invasion of hyphae of the different Scedosporium species in the kidney and spleen during the first days of infection, while no fungal structures were observed at 15 days post-infection in both tissues. In contrast to that shown in the brain of infected mice where increased invasion of Scedosporium hyphae was evidenced in the brain parenchyma during infection, particularly the brain of mice infected with S. apiospermum, S. aurantiacum and S. dehoogii abundant fungal structures were observed during infection.

In conclusion, our results correlate with clinical and experimental findings where the ability of three species S. apiospermum, S. aurantiacum and S. dehoogii as the most pathogenic species with clinical manifestations and fatal prognosis has been evidenced, as well as the ability of these species to disseminate to the brain causing lethal clinical entities. Therefore, further studies are needed to understand scedosporiosis and develop new strategies for the treatment, prevention and prognosis of this mycosis.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Elizondo-Zertuche: Conceptualization, Methodology, Formal analysis, Investigation. Flores-Maldonado: Formal analysis, Visualization, Writing-original draft, Writing-review & editing. Gonzalez G: Conceptualization, Methodology, Validation, Resources, Supervision, Project administration, Funding acquisition. Becerril-Garcia: Formal analysis, Visualization, Investigation, Writing-original draft, Writing-review & editing.

Funding

This work was supported by the Consejo Nacional de Ciencia y Tecnología [CONACyT-INFRA-2015–01-251142].

Declarations

Conflict of interest

The authors declare that they have no conflict of interest. The authors alone are responsible for the content and the writing of the article.