A powerful in vivo alternative model in scientific research: Galleria mellonella

Murine models are suggested as the gold standard for scientific research, but they have many limitations of ethical and logistical concern. Then, the alternative host models have been developed to use in many aspects especially in invertebrate animals. These models are selected for many areas of research including genetics, physiology, biochemistry, evolution, disease, neurobiology, and behavior. During the past decade, Galleria mellonella has been used for several medical and scientific researches focusing on human pathogens. This model commonly used their larvae stage due to their easy to use, non-essential special tools or special technique, inexpensive, short life span, and no specific ethical requirement. Moreover, their innate immune response close similarly to mammals, which correlate with murine immunity. In this review, not only the current knowledge of characteristics and immune response of G. mellonella, and the practical use of these larvae in medical mycology research have been presented, but also the better understanding of their limitations has been provided

Genotyping, antifungal susceptibility testing, and biofilm formation of Trichosporon spp. isolated from urine samples in a University Hospital in Bangkok, Thailand

The basidiomycetes yeast Trichosporon is widespread in the natural environment, but can cause disease, mainly in immunocompromised patients. However, there have been only few studies about this infection in Thailand. In this study, we characterized 53 Trichosporon spp. isolated from urine samples from patients admitted to a single hospital in Bangkok, Thailand over a one-year period from 2019 to 2020. The strains were identified using colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and nucleotide sequence analysis of intergenic spacer 1 (IGS1). Fifty-one isolates were Trichosporon asahii, and the remaining isolates were Trichosporon inkin and other Trichosporon species. Three genotypes of IGS1-1, 3, and 7 were observed among T. asahii. The sensitivity of the yeasts to the antifungal drugs amphotericin B, fluconazole, and voriconazole ranged from 0.25 to >16 jig ml-1, 0.5-8 jig ml-1, and 0.01-0.25 jig ml-1, respectively. We investigated biofilm formation by the isolates, and no biofilm production was found in one isolate, low biofilm production in forty-four isolates, and medium biofilm production in six isolates. T. inkin produced biofilms at low levels, and Trichosporon spp. produced biofilms at medium levels. This research increases our understanding of the molecular epidemiology of Trichosporon spp. isolated from one university hospital in Bangkok, Thailand, and reveals their genetic diversity, antifungal susceptibility profiles, and capacity for in vitro biofilm production

Genetic variation analysis and relationships among environmental strains of Scedosporium apiospermum sensu stricto in Bangkok, Thailand.

The Scedosporium apiospermum species complex is an emerging filamentous fungi that has been isolated from environment. It can cause a wide range of infections in both immunocompetent and immunocompromised individuals. We aimed to study the genetic variation and relationships between 48 strains of S. apiospermum sensu stricto isolated from soil in Bangkok, Thailand. For PCR, sequencing and phylogenetic analysis, we used the following genes: actin; calmodulin exons 3 and 4; the second largest subunit of the RNA polymerase II; ß-tubulin exon 2-4; manganese superoxide dismutase; internal transcribed spacer; transcription elongation factor 1α; and beta-tubulin exons 5 and 6. The present study is the first phylogenetic analysis of relationships among S. apiospermum sensu stricto in Thailand and South-east Asia. This result provides useful information for future epidemiological study and may be correlated to clinical manifestation

List of primers used in the present study.

<p>List of primers used in the present study.</p

Effects of UVC Irradiation on Growth and Apoptosis of Scedosporium apiospermum and Lomentospora prolificans

Scedosporium apiospermum and Lomentospora prolificans are important fungal species isolated from immunocompromised patients. Previous studies have demonstrated that these filamentous fungi exist as saprophytes in the soil and showed the highest minimum inhibitory concentration to several drugs. We aimed to examine how UVC affects the S. apiospermum and L. prolificans by investigating the role of UVC on growth, induction of apoptosis by ethidium bromide (EB)/acridine orange (AO) staining, and transcriptomic study of caspase recruitment domain family, member 9 (CARD-9) gene. Our studies showed that 15 minutes of exposure to UVC light effectively increased reduction in both organisms and caused changes in colony morphology, color, and hyphal growth pattern. After 15 min of UVC irradiation, apoptotic cells were quantitated by EB/AO staining, and the percentage of apoptosis was 96.06% in S. apiospermum and 28.30% in L. prolificans. CARD-9 gene expression results confirmed that apoptosis was induced in S. apiospermum and L. prolificans after UVC treatment and that S. apiospermum showed a higher expression of apoptosis signaling than L. prolificans. Our study explored the effects of UVC in the inactivation of S. apiospermum and L. prolificans. We hope that our data is useful to other researchers in future studies

Molecular phylogenetic maximum likelihood analysis of the concatenated sequences of ACT, CAL, RPB2, BT2 and SOD2 genes.

<p>The tree with the highest log likelihood (-1105.3764) is shown. The black circle represents the strains from Thailand (environmental isolates), the blue circle represents the strains from France (clinical isolates), the green circle represents the strains from China (clinical isolates) and the pink circle represents the strains from Japan (clinical isolates).</p

Allele types (ATs) and sequence types (STs) of ACT, CAL, RPB2, BT2, SOD2, ITS, TEF-1α, and TUB genes and STs.

<p>Allele types (ATs) and sequence types (STs) of ACT, CAL, RPB2, BT2, SOD2, ITS, TEF-1α, and TUB genes and STs.</p

Distribution of Scedosporium species in soil from areas with high human population density and tourist popularity in six geographic regions in Thailand.

Scedosporium is a genus comprising at least 10 species of airborne fungi (saprobes) that survive and grow on decaying organic matter. These fungi are found in high density in human-affected areas such as sewage-contaminated water, and five species, namely Scedosporium apiospermum, S. boydii, S. aurantiacum, S. dehoogii, and S. minutisporum, cause human infections. Thailand is a popular travel destination in the world, with many attractions present in densely populated areas; thus, large numbers of people may be exposed to pathogens present in these areas. We conducted a comprehensive survey of Scedosporium species in 350 soil samples obtained from 35 sites of high human population density and tourist popularity distributed over 23 provinces and six geographic regions of Thailand. Soil suspensions of each sample were inoculated on three plates of Scedo-Select III medium to isolate Scedosporium species. In total, 191 Scedosporium colonies were isolated from four provinces. The species were then identified using PCR and sequencing of the beta-tubulin (BT2) gene. Of the 191 isolates, 188 were S. apiospermum, one was S. dehoogii, and species of two could not be exactly identified. Genetic diversity analysis revealed high haplotype diversity of S. apiospermum. Soil is a major ecological niche for Scedosporium and may contain S. apiospermum populations with high genetic diversity. This study of Scedosporium distribution might encourage health care providers to consider Scedosporium infection in their patients

ผลของอาหารเลี้ยงเชื้อต่อการเจริญเติบโตและสัณฐานวิทยาของเชื้อ Scedosporium boydii และ Scedosporium prolificans The influence of culture media on growth and morphology of Scedosporium boydii and Scedosporium prolificans

 บทคัดย่อ อาหารเลี้ยงเชื้อที่ใช้ในการเพาะเลี้ยงเชื้อแต่ละชนิดจะมีความจำเพาะของสารอาหารแตกต่างกันออกไปขึ้นอยู่กับความต้องการของเชื้อ การวิจัยในครั้งนี้มีวัตถุประสงค์เพื่อศึกษาการเจริญเติบโตและสัณฐานวิทยาของเชื้อ Scedosporium boydii และ Scedosporium prolificans ซึ่งจะศึกษาระหว่าง selective media และอาหารเลี้ยงเชื้อพื้นฐานที่มีการเติมสารอาหารเพิ่มลงไป ซึ่งทำการศึกษาโดยการใส่อาหารเลี้ยงเชื้อและเชื้อแต่ละชนิดในถาดเลี้ยงเชื้อชนิด 6 หลุม แล้วนำไปบ่มที่อุณหภูมิ 37 องศาเซลเซียสเป็นเวลา 4 วัน จากนั้นวัดอัตราการเจริญเติบโตโดยการวัดเส้นผ่าศูนย์กลางของโคโลนี และศึกษาสัณฐานวิทยาของ S. boydii และ S. prolificans จากการศึกษาพบว่า เชื้อ S. boydii และ S. prolificans ที่เลี้ยงบนอาหารเลี้ยงเชื้อ SDA+10%FBS มีการเจริญเติบโตในแต่ละวันมากที่สุด และเมื่อวัดเส้นผ่าศูนย์กลางของโคโลนีใน วันที่ 4 ได้เท่ากับ 31.67± 1.53 และ 25.33± 2.36 มิลลิเมตร และมีอัตราการเจริญเติบโตคือ 7.92 และ 6.33 มิลลิเมตรต่อวัน ตามลำดับ สำหรับการศึกษาลักษณะสัณฐานวิทยาจากการสังเกตโคโลนีด้วยตาเปล่า พบว่าโคโลนีของเชื้อ S. boydii เจริญแผ่กว้าง มีสีขาว ปุย และค่อยๆ เปลี่ยนเป็นสีเข้มขึ้น ส่วนเชื้อ S. prolificans จะมีลักษณะโคโลนีคล้ายกันคือ เจริญแผ่กว้าง มีสีเทาอ่อน ปนขาว  เมื่อเพาะเลี้ยงนานขึ้น โคโลนีจะมีสีเข้มขึ้นจะเป็นสีดำ ในส่วนของลักษณะทางกล้องจุลทรรศน์เมื่อย้อมด้วย Lactophenol cotton blue พบว่าลักษณะโคนิเดีย ของแต่ละเชื้อในอาหารที่ต่างกันนั้นไม่มีความแตกต่างกัน คือ มีรูปร่างทรงกลมถึงค่อนข้างกลม ดังนั้นจึงสรุปได้ว่า อาหารเลี้ยงเชื้อ SDA+10%FBS เหมาะสมที่จะใช้เป็นทางเลือกในการนำมาเพาะเลี้ยงเชื้อ S. boydii และ S. prolificans เพื่อให้เชื้อมีการเจริญเติบโตรวดเร็วขึ้น Abstract The culture media for fungal cultivation contained several specific and various nutrients in order to support the growth of fungal. This study aimed to determine growth and morphology of Scedosporium boydii and Scedosporium prolificans on different types of culture media. The selective media and standard nutrient added media were used to study the growth rate and morphology of S. boydii and S. prolificans by adding each medium into 6 well plates. Each type of medium was used to culture both S. boydii and S. prolificans and incubated at 37 degree Celsius condition for 4 days. The growth rate was determined by colonies diameter measurement. Morphological analysis was determined in S. boydii and S. prolificans in different types of culture medium. The results showed that colonies of S. boydii and S. prolificans on SDA+10%FBS exhibited larger size than colonies growing on other culture media on the fourth day of cultivation (31.67 ± 1.53 and 25.33± 2.36 mm respectively). The growth rate of S. boydii and S. prolificans was 7.92 and 6.33 mm/day respectively. The morphological study of S. boydii colony showed wide expanded, white color, and gradually changed to gray color. S. prolificans colony showed wide expanded, gray-white color, and gradually changed to gray and black color. In addition, the lactophenol cotton blue stain showed no difference in conidia shape of both S. boydii and S. prolificans (globose-subglobose shape). Therefore, this study demonstrated that SDA+10%FBS represented proper and alternative medium choice of S. boydii and S. prolificans culture for rapid growth culture system