Ym broth

Lactiplantibacillusplantarum cell‐free culture supernatants (CFCS) were prepared from the spent media collected after L. plantarum incubation in cMRS for 24 h at 30°C. CFCS was collected by centrifugation at 4000× g for 10 min at 4°C followed by filtration of the supernatant through a 0.45 μm polyethersulfone (PES) filter (Genesee Scientific, San Diego, CA). To eliminate the effects of differences of pH on yeast inhibition, the CFCS was adjusted with lactic acid (1.3 M) to pH 3.8, the lowest pH reached by L. plantarum after incubation in cMRS (data not shown). S. cerevisiae UCDFST 09‐448 (Golomb et al., 2013 (link)), a strain shown to cause olive tissue damage and spoilage during olive fermentations, was grown in yeast mould (YM) broth (BD, Franklin Lakes, NJ) for 24 h at 30°C with aeration at 250 rpm. Cells were collected by centrifugation at 20 000× g for 5 min at 4°C and then washed twice with PBS. S. cerevisiae UCDFST 09‐448 was then inoculated into 96‐well microtiter plates containing 1:1 ratio of 2X YM and CFCS at a starting OD₆₀₀ of 0.05. OD₆₀₀ was measured in a Synergy 2 microplate reader (Biotek, Winooski, VT) set at 30°C for 24 h aerated every hour by shaking for 10 s before each read. Controls included S. cerevisiae UCDFST 09‐448 incubated in YM and YM supplemented with cMRS (pH 3.8).

Bacterial vaginosis-associated bacteria G. vaginalis (ATCC 14018, ATCC 49145), candidiasis pathogen Candida albicans (ATCC 14053), trichomoniasis pathogen Trichomonas vaginalis (ATCC 30001), and human vagina-derived lactobacilli, Lactobacillus crispatus (ATCC 33820), Lactobacillus gasseri (ATCC 33323), Lactobacillus plantarum (ATCC 14917), and Lactobacillus jensenii (ATCC 25258), were purchased from the American Type Culture Collection (ATCC). Gardnerella vaginalis M^R is a spontaneous metronidazole-resistant mutant of ATCC 14018. The clinical isolates of BV-associated bacteria and healthy human vagina lactobacilli were kindly provided by Dr. Chuang at National Yang Ming Chiao Tung University, Taiwan (all bacterial isolates are listed in Table 1). All BV-associated bacteria were cultured in NYCIII broth (ATCC medium 1685) and grown at 37°C in anaerobic conditions, using AnaeroPack®-Anaero (MGC, Japan). All Lactobacillus spp. were cultured in MRS broth (Difco BD) and grown at 37°C in facultatively anaerobic conditions, using AnaeroPack®-MicroAero (MGC, Japan), except L. gasseri was grown in aerobic conditions. Candida albicans was cultured in YM broth (Difco BD) and grown at 37°C in aerobic conditions.

The morphological and physiological description was performed according to Suh et al. [28 ]; their protocols conform to the standard outlined in The Yeasts [29 ]. Culture morphology was observed on YMA, corn meal agar (CMA), and YM broth (BD; Thermo Fisher). After seven-day incubation, colonies were described in terms of elevation, margin, color (oac; [30 ]), form, and surface texture. Individual cells were examined under a compound microscope (Olympus BH-2; Tokyo, Japan). Micrographs were captured using an Olympus SC30 camera. A total of 30–180 cells were measured per isolate per treatment using Piximètre v5.10 (http://www.piximetre.fr/, accessed on 4 May 2021). Carbon and nitrogen assimilations were performed according to Suh et al. [28 ]. Positive assimilations marked with “+++” or “++” were reassigned as “+” while negative assimilations were assigned as “−”; weak growth was denoted as “(w)”, and delayed growth was denoted as “(d)”. This modified scale was used to normalize our data and allow for comparison across previously published data with varying scales.