Table 1.
Demographic information of study participants.
Figure 1.
Bacterial microbiome (bacteriome) of HIV-infected patients and uninfected individuals.
(A) Relative abundance bacteria in uninfected and HIV-infected patients (N = 11 for each group), (B) Bacteria present in the core oral bacteriome of uninfected and HIV-infected individuals.
Figure 2.
Fungal microbiome (mycobiome) of HIV-infected patients and uninfected individuals.
(A) Relative abundance of fungi in uninfected and HIV-infected patients (N = 12 for each group), (B) Fungi present in the core oral mycobiome of uninfected and HIV-infected individuals.
Figure 3.
Correlation coefficients of abundance of microbes in uninfected and HIV-infected patients.
Correlation of bacteriome and mycobiome was determined for (A) uninfected and (B) HIV-infected study participants using R statistical computing software (Spearman's correlation and two-tailed probability of t for each correlation) for the two groups. Interactions among different fungi in the mycobiome of (C) uninfected and (D) HIV-infected study participants was also determined using similar approach. Red: Positive correlation; Blue: negative correlation; diameter of circles represent the absolute value of correlation for each pair of the microbe-microbe matrix.
Table 2.
Correlation between bacteriome and mycobiome in uninfected and HIV-infected study participants.
Table 3.
Correlation among oral fungi in uninfected study participants.
Figure 4.
Antagonistic relation between Pichia and other fungi.
(A) Relative abundance of Pichia and Candida in uninfected individuals. (B) Effect of PSM on growth of Candida was determined by measuring optical density (OD). Effect of PSM on (C) Aspergillus, and (D) Fusarium was determined by measuring dry weight of fungi. Pichia spent (“conditioned”) medium (PSM) was obtained by centrifuging 100-mL culture of Pichia grown in Sabouraud dextrose broth (SDB).
Table 4.
Relationship between Pichia and other fungi in oral wash of uninfected patients.
Figure 5.
Pichia overgrows Candida in mixed cultures.
Pichia farinosa was mixed with GFP-tagged C. albicans strain at 103 cells each in yeast nitrogen base (YNB) growth medium and incubated at 37°C for 54 h. Growth of Candida cells was monitored by phase-contrast and fluorescence microscopy. Panels A, D, G: phase-contrast images of Candida alone, Candida+Pichia, and Pichia alone, respectively. Panels B, E: fluorescence images of Candida alone and Candida+Pichia mixture. Panels C, F: Composite figures with phase-contrast images merged with fluorescence image, for Candida only and Candida+Pichia mixture of cells. Panel H: quantitative measurement of fluorescence (excitation and emission wavelength = 485 and 435 nm, respectively).
Figure 6.
Activity of Pichia spent medium (PSM) against fungal biofilms.
(A) Effect of Pichia cells on the ability of Candida to form biofilms. Candida and Pichia were co-incubated [Candida∶Pichia (C∶P) = 3∶1, 1∶1, or 1∶3] and biofilm formation was monitored (*P≤.002, compared to Candida or Pichia controls). (B) Effect of media supernatant obtained from Pichia, Penicillium, or Cladosporium on Candida biofilms. Mean ± SD of ≥3 separate experiments. (C–E) Confocal microscopy images of Candida biofilms formed in presence of (C) no media supernatant, (D) Penicillium supernatant or (E) Pichia supernatant. (F) Thickness of biofilms formed in presence of media supernatant of Pichia or Penicillium.
Figure 7.
Dose dependent activity of PSM, and its effect on Candida germination and adhesion.
Effect of undiluted and diluted (50%) PSM on (A) metabolic activity and (B) thickness of Candida biofilms was assessed. (C) Germination in Candida grown in SDB, exposed to fetal bovine serum, (D) Stunted germ tubes formed by Candida exposed to Pichia supernatant (Magnification 20X), (E) Effect of PSM on the ability of Candida (grown in SDB) to adhere to solid substrates. (SDB - Sabouraud dextrose broth.)
Figure 8.
Biochemical characterization of Pichia spent medium.
(A) Metabolic activity of Candida biofilms exposed to metabolites extracted from spent media of Pichia, Penicillium, or media control. (B) Effect of PSM exposed to proteinase, heat, or alkali on Candida biofilms. Confocal images show architecture of Candida biofilms exposed to (C) no PSM (control), (D) untreated PSM, (E) proteinase-K treated PSM, (F) heat, or (G) alkali.
Figure 9.
Efficacy of Pichia spent medium (PSM) in an experimental murine morel of oral candidiasis.
Assessment of oral candidiasis in mice infected with Candida was performed by (A) clinical score and (B) tongue fungal burden. (A) Median clinical scores of oral candidiasis in mice after treatment with PSM and nystatin. (B) Box-plot of tissue fungal burden (log CFUs/g) in different groups of mice infected with Candida (P-values, compared to PSM treatment). Histology analyses of tissue section of tongue from mouse infected with Candida, followed by (C) no treatment or treated with (D) vehicle control, (E) nystatin, or (F) PSM. Arrows – fungal hyphae.