Growth Inhibition of an Opportunistic Yeast Pathogen Trichosporon asahii by Staphylococcus epidermidis

Abstract

In the co-culture of Staphylococcus epidermidis and Trichosporon asahii, a fungal pathogen, it was observed that live S. epidermidis inhibited the growth of T. asahii. Soluble active anti-T. asahii substances were speculated to be produced by S. epidermidis in culture medium. Using ¹H- and ¹³C-NMR spectra and electron ionization-high resolution mass spectrometry (HR-negative-FAB-MS), we separated the active molecule and identified it as lactic acid. Commercially available L-lactic acid and D-lactic acid inhibited the growth of T. asahii. These results show that metabolites from bacterial populations are involved in the interactions of pathogenic fungi. The use of antibacterial agents to treat primary diseases could lead to the disruption of normal microbial communities and could cause opportunistic infections such as trichosporonosis.

Interactions between living things in nature, including microorganisms that live inside the bodies of animals, may cause coexistence or exclusion.¹⁾ Focusing on the interactions between bacteria and fungi, we found that Staphylococcus aureus adhered to the cells of Cryptococcus neoformans, a fungal pathogen, inducing its death. Triosephosphate isomerase, a glycolytic enzyme, on the cell surface of S. aureus and mannose residues in the capsule surrounding C. neoformans were identified as the adhesion molecules.^2–6) By investigating other combinations of microbes that threaten human health, we noticed that Trichosporon asahii did not grow in co-cultures with S. epidermidis using solid medium. Therefore, we speculated that the active substances might be soluble.

T. asahii is a yeast-like fungus found in the environment (e.g., in decaying logs and soil) and is also a pathogen of opportunistic infection. Trichosporonosis is a life-threatening mycosis, particularly for patients with hematological malignancies with a poor prognosis.^7–9) T. asahii is also known as an antigen for summer-type hypersensitivity pneumonitis (SHP). In Japan, SHP is considered a type III or IV allergy that occurs mainly in summer from April to November.¹⁰⁾ Most cases live in wooden houses of 30 years old. Ando et al.^11,12) isolated T. asahii from the environment of such cases, and detected anti-T. asahii antibodies in 99.2% of those examined. Using a nested PCR assay, Zhang et al.¹³⁾ showed that T. asahii colonized the skin of healthy humans and the detection frequency increased with age. Cho et al.¹⁴⁾ suggested that T. asahii formed a part of the normal fungal microbiota of the gut in humans based on ribosomal RNA genotypes. The strains detected in the human body showed different genotypes from those isolated from the environment, suggesting that T. asahii may cause trichosporonosis.^15,16)

S. epidermidis is a well-known species and is a normal component of the bacterial microbiota in humans.¹⁷⁾ This common bacterium could play a role in the defense of pathogens including T. asahii. We investigated the mechanism underlying the growth inhibition of T. asahii by S. epidermidis.

MATERIALS AND METHODS

Growth Inhibition Tests by S. epidermidis

T. asahii CBS2479 (type strain of T. asahii AKAGI ex SUGITA et al.) was cultured in Sabouraud broth (0.5% yeast extract, Becton, Dickinson and Company, U.S.A.; 1% polypeptone, Nihon Seiyaku, Japan; 2% glucose, Yoshida Seiyaku, Japan) for 2 d at 27°C. The culture was diluted with physiological saline solution (PSS) to 100-fold and 1 mL was placed into a Petri dish. About 20 mL warm Sabouraud agar (yeast extract 0.5%, polypeptone 1%, glucose 2%, agar, Wako Pure Chemical Industries, Ltd., Japan 1.5%) was poured into the dish and mixed well. After solidification, a well was made in the agar medium containing T. asahii using a penicillin cup (outer diameter 8 mm, inner diameter 6 mm, height 10 mm), and 100 µL S. epidermidis NBRC 100911 (type strain) live cell culture or extract from S. epidermidis was poured into the well and incubated at 37°C.

Preparation of Anti-T. asahii Fractions from Culture Medium of S. epidermidis

S. epidermidis was cultured in 600 mL Tripticase soy broth (Becton, Dickinson and Company) supplemented with glucose to a final concentration of 2% for 2 d at 37°C with shaking. Cells were removed by centrifugation at 4500 rpm for 10 min and filtered with a membrane of pore size 0.45 µm. A mixture of the culture supernatant and n-butanol in equal volumes was shaken in a separating funnel for 10 min and the n-butanol layer was collected and concentrated with an evaporator to about 10 mL. The n-butanol extract was fractionated by silica gel column chromatography (diameter of 2.0 cm, length of 45 cm) using a stepwise elution of 100, 75, 50, 25, and 0% ethanol. The fraction, which was confirmed to inhibit the growth of T. asahii, was concentrated and subjected to a reverse-phase cartridge column (Sep-Pack C18, Waters, U.S.A.) using a stepwise elution of 0, 25, 50, 75, and 100% acetonitrile containing 0.1% trifluoroacetic acid (TFA). The active fraction in the pretreatment column was concentrated and further purified using a reverse-phase HPLC column (InertSustain, RP-C18, 4.6×250 mm) to identify the compound that inhibited the growth of T. asahii. The peak associated with antifungal activity was collected, concentrated to dryness under a vacuum, and then subjected to ¹H- and ¹³C-NMR spectral analyses and high resolution (HR)-negative-FAB-MS analysis to determine the structure of the antifungal component. ¹H- and ¹³C-NMR spectra were recorded using a JEOL JMN-Lambda-500 spectrometer (¹H: 500 MHz; ¹³C: 125 MHz). The chemical shifts are reported as δ values relative to the signal for the trimethylsilyl group in 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) at 0 ppm. HR-FAB-MS was performed with a JEOL JMS-700 double-focusing spectrometer.

Lactic Acid Determination

S. epidermidis was cultured in Sabouraud broth for 2 d. The cell-free culture supernatant was isolated at 24 and 48 h by centrifugation (3500 rpm, 5 min) paired with filtration (0.45 µm). The supernatant was then used for determination of lactic acid. Lactic acid was measured using an HPLC method¹⁸⁾ with minor modifications. Briefly, the supernatant (5 µL) was injected onto an ODS-4 column (4.0×250 mm, GL Science, Japan), using 0.1% formic acid as the mobile phase at a flow rate of 0.8 mL/min. The eluent was monitored by UV-210 nm. L-Lactic acid was used as the standard.

RESULTS

Growth Inhibition of T. asahii by S. epidermidis

The effects of S. epidermidis on the growth of T. asahii were examined using Sabouraud agar containing live cells of T. asahii. The well was bored and 100 µL of S. epidermidis culture was grown in nutrient broth at 37°C for 2 d. As shown in Fig. 1A, an inhibitory zone of T. asahii was observed near the well containing S. epidermidis. When using Sabouraud agar containing phenol red, the inhibitory zone was determined to have an acidic pH (Fig. 1B). On the other hand, S. epidermidis did not inhibit the growth of C. neoformans B-3501 or C. albicans CBS562 under the same conditions (Figs. 1C, D).

Fig. 1. Growth Inhibition of T. asahii by S. epidermidis

Live yeast cells were mixed with warm Sabouraud agar. After making a well in the agar medium, live S. epidermidis cells were poured into the well and incubated at 37°C. Photographs show the results of T. asahii (A), T. asahii using medium containing phenol red (B), C. neoformans (C), and C. albicans (D).

Fractionation and Identification of the Anti-T. asahii Substance

We speculated that the unidentified substances inhibiting the growth of T. asahii may be water-soluble molecules. Cell-free supernatant of S. epidermidis culture was shaken with n-butanol, and the extract was concentrated to perform a growth inhibition test. As inhibition of T. asahii growth was observed, the extract was applied to column chromatography. The fraction that was eluted by approximately 50% ethanol during silica gel column chromatography showed growth inhibition (Fig. 2A) because T. asahii did not grow near the well containing this fraction. The fraction was separated using a reverse-phase cartridge column and the sample eluted with water exhibited inhibitory activity (Fig. 2B). This crude active fraction was further purified using a reverse-phase (C₁₈) HPLC column with UV detection. As shown in Fig. 3A, many peaks were observed on the chromatogram; however, anti-T. asahii activity was only observed for a major hydrophilic peak (Peak 1) (Fig. 3B). Peak 1 was subjected to ¹H- and ¹³C-NMR spectra and HR-negative-FAB-MS analysis, and lactic acid was identified as an antifungal component.

Fig. 2. Determination of Anti-growth Activities of Fractions in Each Step

Photographs show the results using the fraction eluted with 50% ethanol in silica gel column chromatography (A), eluted with water on a C₁₈ cartridge column (B).

Fig. 3. A Chromatogram of the Crude Hydrophilic Fraction Exhibiting Antifungal Activity

To identify the antifungal compound, we separated and fractionated three peaks, P1–P3 (A), and tested their growth inhibitory activities (B). The fractions that were eluted from about minute 28 onward did not exhibit antifungal activity.

Characterization Data

Isolated lactic acid: ¹H-NMR (D₂O) δ: 1.36 (3H d, J=6.8 Hz, –CH₃), 4.33 (1H, d, J=6.8 Hz, –CH(OH)–). ¹³C-NMR (D₂O) δ: 22.0 (q), 69.3 (d), 181.3 (s). HR-negative-FAB-MS (glycerol): m/z 89.0237 [M⁻−H] (Calcd for C₃H₅O₃: 89.0239). These data were identical to those of authentic L-lactic acid.

Growth Inhibition Tests by Lactic Acid

Because lactic acid was identified as the anti-T. asahii active agent produced by S. epidermidis, growth inhibition tests were performed using commercially available L-lactic acid and D-lactic acid. As shown in Fig. 4, growth of T. asahii was inhibited by both undiluted (approximately 12 M) and 5-fold diluted lactic acid. Live S. epidermidis did not inhibit the growth of C. neoformans and C. albicans (Fig. 1), but lactic acid reagents did inhibit their growth, as shown in Figs. 5 and 6. However, the inhibitory zone was smaller in the case of C. neoformans and C. albicans.

Fig. 4. Growth Inhibition of T. asahii by L-Lactic Acid and D-Lactic Acid, Using Undiluted and 5-Fold Diluted Lactic Acid

One hundred microliters of lactic acid was poured into the well.

Fig. 5. Growth Inhibition of C. neoformans by L-Lactic Acid and D-Lactic Acid, Using Undiluted and 5-Fold Diluted Lactic Acid

One hundred microliters of lactic acid was poured into the well.

Fig. 6. Growth Inhibition of C. albicans by L-Lactic Acid and D-Lactic Acid, Using Undiluted and 5-Fold Diluted Lactic Acid

One hundred microliters of lactic acid was poured into the well.

Determination of Lactic Acid in Culture Medium

Lactic acid determination was performed using an HPLC-based method. The peak identified as lactic acid was not detected in Sabouraud broth before inoculation of S. epidermidis, but was detected in the culture supernatant after incubation. The amounts of lactic acid present after 24 and 48 h of incubation were calculated as 1.1 mg/mL (12.2 mM) and 2.4 mg/mL (26.6 mM), respectively.

DISCUSSION

Bacterial metabolites can be both useful and harmful to other living things.^19–23) We purified and analyzed the molecule produced by S. epidermidis that inhibited an opportunistic fungal pathogen, T. asahii, and identified it as lactic acid. We attempted quantitative analysis of lactic acid in the culture supernatant of S. epidermidis by HPLC. The contents in the medium were the same as in the inhibition assay, but without agar. In the medium prior to inoculation of S. epidermidis, lactic acid was not found, but it was determined and gradually accumulated in culture mediums along with the growth of S. epidermidis for 48 h. The growth inhibition zone of T. asahii was near living S. epidermidis. Lactic acid diffused in agar medium and inhibited growth at concentrations higher than required.

C. albicans and C. neoformans were less sensitive than T. asahii. Accumulation of D-lactate and L-lactate has been reported based on the pyruvate metabolism of S. epidermidis (http://www.genome.jp/kegg-bin/show_pathway?ser00620). In our experiments, lactic acid was identified and both L-lactic acid and D-lactic acid, which are commercially available, inhibited the growth of T. asahii. We observed that S. epidermidis acidified the growth medium, but T. asahii alkalinized it. When the concentration of glucose in the agar medium was varied by 0.25, 0.5, and 1.0%, growth inhibition was observed with 1.0% glucose, but T. asahii grew well at 0.25 and 0.5% glucose in proximity to S. epidermidis. These findings support the hypothesis that the compound inhibiting the growth of T. asahii is related to glycolysis and pyruvate metabolism with lactic acid release. Matsuda et al.²⁴⁾ reported the antimicrobial activities of organic acids using 15 strains of bacteria, 6 strains of yeast, and 2 strains of molds. L-Lactic acid and D-lactic acid did not have inhibitory activities against fungi, but T. asahii was not tested. The fungi used in the study were ascomycetous yeasts, which are taxonomically distant from Trichosporon, a genus of basidiomycetous yeasts.²⁵⁾ Recently, Trzaska et al. reported that acetic acid is highly effective against major pathogenic groups of Mucorales.²⁶⁾ This antifungal effect is not observed with other acids, such as hydrochloric and lactic acid, suggesting that acetic acid activity against Mucorales spores is not solely evoked by low environmental pH. We performed the experiments using hydrochloric acid. T. asahii grew well in the presence of 0.1 M HCl. This may be due to low permeability across the membrane, as the concentration of undissociated acid was lower when using HCl. According to the pyruvate metabolism of S. epidermidis, acetic acid might accumulate in addition to lactic acid. Therefore, the anti-T. asahii activity of acetic acid was tested and we observed that acetic acid inhibited the growth of T. asahii as well as C. neoformans and C. albicans. Here, we identified lactic acid in the active fraction prepared from the culture supernatant of S. epidermidis. It is possible that acetic acid is involved in the inhibition of T. asahii proliferation. In the human body, prevention of overgrowth of T. asahii by S. epidermidis on the skin may be possible. However, in the intestinal tract, lactic acid might be generated by lactic acid bacteria. Since lactic acid was identified as an inhibitor in this study, such bacteria would have the ability to control yeast pathogens.

The persistence of S. epidermidis as part of a normal bacterial microbiota would be helpful for defense against pathogens that cause opportunistic infection. Therefore, the use of antibacterial agents used in primary diseases could lead to the disruption of normal microbial communities.

Conflict of Interest

The authors declare no conflict of interest.

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