P364 Quantitative monitoring of Aspergillus fumigatus mycelial growth by optical density

Abstract   Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi from applying the growth monitoring methods used for unicellular organisms such as yeast and bacteria. We have analyzed the biological functions of cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) in Aspergillus species, and revealed that both α-1,3-glucan and GAG contribute to the pellet formation. Here we constructed the double disruption mutant of α-1,3-glucan and GAG biosynthesis-related genes (Δags1Δgtb3) in Aspergillus fumigatus AfS35 strain, and used the mutant for quantitative monitoring of the mycelial growth by optical density. Methods To measure the optical density of conidia and mycelia in shake-flask culture, conidia (final concentration, 1.0 × 10⁷/ml) of AfS35 or Δags1Δgtb3 strain were inoculated into 50 ml of Aspergillus minimal medium (AMM) medium in a 200-ml Erlenmeyer flask and rotated at 160 rpm at 37°C. At each sampling point, the culture (2 ml) was withdrawn, and 100 μL of the culture was mixed with 100 μL of 4% paraformaldehyde solution in a 96-well plate. OD₆₀₀ was measured in a microplate reader. To evaluate the susceptibility of the Δags1Δgtb3 strain to antifungal agents, conidia (final concentration, 5.0 × 10⁶/ml) were inoculated into 500 μL of RPMI medium containing an antifungal agent (voriconazole, itraconazole, amphotericin B, flucytosine, or micafungin) in a 48-well plate and rotated at 300 rpm using a microplate mixer at 35°C for 15 h, and OD₆₀₀ was measured in triplicate. Results In AMM flask culture, pellets became visible in AfS35 from 9 h, and their size increased with time, whereas the Δags1Δgtb3 hyphae were continuously dispersed (Fig. 1a). The OD₆₀₀ measurements of AfS35 suggested no correlation between apparent AfS35 growth and OD₆₀₀ (Fig. 1b). In the Δags1Δgtb3 strain, the first and third quartiles were 0.633 and 0.595 at 15 h (Fig. 1b), suggesting that the measurement of OD₆₀₀ is suitable for monitoring the growth of the mutant. As an application of the monitoring method, the Δags1Δgtb3 strain was grown in an RPMI medium containing the indicating antifungal agent for 15 h in a 48-well plate (Fig. 2). Growth was repressed in the presence of antifungal agents tested, except for micafungin (Fig. 2). Growth was completely inhibited at 2 μg/ml of voriconazole, 1 μg/ml of itraconazole, 0.5 μg/ml of amphotericin B, and 256 μg/ml of flucytosine (Fig. 2), which was in agreement with MICs determined by CLSI M38-A2. Conclusion We established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly applied to screening for novel antifungals against Aspergillus species.

Results: A total of 101 fungal species were isolated from water samples. Yeasts 48 ( 47.5% ) , Rhodotorula 34 ( 33.6% ) accounted for the unicellular fungi. Among Filamentous mycoflora, the most common isolated fungi were Aspergillus species 13 ( 12.8% ) , followed by Fusarium spp 4 ( 3.9% ) with Mucor and | Rhizopus species accounting for 1 ( 0.9% ) respectively. Yeasts species were the most common species isolated from water species and Aspergillus species were more frequently isolated from filamentous fungi.
Conclusion: Yeasts species were the most common species isolated from water species and the roles they play in biofilm needs to be further investigated.

Quantitative monitoring of Aspergillus fumigatus mycelial growth by optical density
Ken Miyazawa 1 , Takashi Umeyama 1 , Yasutaka Hoshino 1 , Shogo Takatsuka 1 , Yasunori Muraosa 1 , Keietsu Abe 2 , Yoshitsugu Miyazaki 1 1 National Institute of Infectious Diseases, Tokyo, Japan 2 Tohoku University, Sendai, Japan Poster session 3, September 23, 2022, 12:30 PM -1:30 PM Objectives: Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi from applying the growth monitoring methods used for unicellular organisms such as yeast and bacteria. We have analyzed the biological functions of cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan ( GAG ) in Aspergillus species, and revealed that both α-1,3-glucan and GAG contribute to the pellet formation. Here we constructed the double disruption mutant of α-1,3-glucan and GAG biosynthesis-related genes ( ags1 gtb3 ) in Aspergillus fumigatus AfS35 strain, and used the mutant for quantitative monitoring of the mycelial growth by optical density.
Methods: To measure the optical density of conidia and mycelia in shake-flask culture, conidia ( final concentration, 1.0 × 10 7 /ml ) of AfS35 or ags1 gtb3 strain were inoculated into 50 ml of Aspergillus minimal medium ( AMM ) medium in a 200-ml Erlenmeyer flask and rotated at 160 rpm at 37°C. At each sampling point, the culture ( 2 ml ) was withdrawn, and 100 μL of the culture was mixed with 100 μL of 4% paraformaldehyde solution in a 96-well plate. OD 6 n n was measured in a microplate reader. To evaluate the susceptibility of the ags1 gtb3 strain to antifungal agents, conidia ( final concentration, 5.0 × 10 6 /ml ) were inoculated into 500 μL of RPMI medium containing an antifungal agent ( voriconazole, itraconazole, amphotericin B, flucytosine, or micafungin ) in a 48-well plate and rotated at 300 rpm using a microplate mixer at 35°C for 15 h, and OD 6 n n was measured in triplicate.
Results: In AMM flask culture, pellets became visible in AfS35 from 9 h, and their size increased with time, whereas the ags1 gtb3 hyphae were continuously dispersed ( Fig. 1a ) . The OD 6 n n measurements of AfS35 suggested no correlation between apparent AfS35 growth and OD 6 n n ( Fig. 1b ) . In the ags1 gtb3 strain, the first and third quartiles were 0.633 and 0.595 at 15 h ( Fig. 1b ) , suggesting that the measurement of OD 6 n n is suitable for monitoring the growth of the mutant. As an application of the monitoring method, the ags1 gtb3 strain was grown in an RPMI medium containing the indicating antifungal agent for 15 h in a 48-well plate ( Fig. 2 ) . Growth was repressed in the presence of antifungal agents tested, except for micafungin ( Fig. 2 ) . Growth was completely inhibited at 2 μg/ml of voriconazole, 1 μg/ml of itraconazole, 0.5 μg/ml of amphotericin B, and 256 μg/ml of flucytosine ( Fig. 2 ) , which was in agreement with MICs determined by CLSI M38-A2.
Conclusion: We established a convenient strategy to monitor A . fumigatus hyphal growth. Our method can be directly applied to screening for novel antifungals against Aspergillus species.