Yeast's vacuole a privileged niche that protects intracellular bacteria against antibiotics

Detection of Helicobacter pylori, Staphylococcus, Nocardia and Cyanobacteria inside the yeast Candida tropicalis raised the question whether treating yeast with antibiotics mix (ABM) eliminates intracellular bacteria. Live/Dead staining showed occurrence of viable bacteria inside the vacuole of C. tropicalis. Amplification of bacterial 16S rRNA genes from yeast DNA with the size of 521, 750, 606 and 450 bp were similar to those from control H. pylori, Staphylococcus, Nocardia and Cyanobacteria, respectively. To eliminate intracellular bacteria yeast cultures in yeast-glucose (YG) broth were treated with 32-1024 μg/mL of ABM (amoxicillin, ciprofloxacin, rifampin and metronidazole) for up to 24 h. Viability of treated yeast cells and their intracellular bacteria was assessed by colony count, Live/Dead staining and detection of bacterial 16S rRNA genes. Colony count of C. tropicalis exposed to 32-256 μg/mL of ABM (4.39-9.63) or 512-1024 μg/mL (9.67-9.77) were similar to their respected controls (p > 0.05). Amplification of similar bacterial genes from treated yeasts and controls confirmed persistent occurrence of intracellular bacteria. Micrographs of yeasts treated with 32-256 μg/mL of ABM showed intact yeasts and intracellular bacteria, however those treated with 512 and 1024 μg/mL showed occurrence of < 10% and > 10% yellow damaged yeasts, respectively that accumulated yellow rifampin. Fluorescence microscopy showed that both intact and damaged yeasts carried live bacteria inside their vacuole. Culture of treated yeasts on YG agar produced colonies with totally intact yeasts and intracellular bacteria. Yeast extruded antibiotics and reduced their effective concentration for killing intracellular bacteria. Establishment of bacteria inside the fungal vacuole cannot be disrupted with antibiotics.