# Nutrient Deficiency Promotes the Entry of _Helicobacter pylori_ Cells into _Candida_ Yeast Cells
## Results
### Search for BLBs within Yeasts of the Genus Candida  
In wet mounts of _H. pylori-Candida_ co-cultures observed using the 100X objective lens of an optical microscope, the presence of BLBs within the vacuoles of yeast (Figure 5A, black arrows) was detected. The movement of BLBs within the vacuoles is shown in Video S1. In addition, we observed an accumulation of coccoid and bacillary bacteria on the yeast pseudohyphae previously co-cultured with _H. pylori_ cells (Figure 5A, red arrow). Cells from a pure _Candida_ culture showed, as expected, the absence of BLBs (Figure 5B). When counting the Y-BLBs in the different co-cultures in BB supplemented with 1%, 5% or 20% FBS, the highest percentage of Y-BLBs (53% to 60%) was obtained in the co-cultures where 1% FBS was used, followed by co-cultures supplemented with 5% and 20% FBS (22% to 26% and 7% to 12%, respectively) (Figure 6). On the other hand, the lowest Y-BLBs percentages were observed in the co-cultures incubated in SS-only (Figure 6). Figure 3: Growth curves of different _H. pylori_ strains in the presence of BB supplemented with 1%, 5% or 20% FBS or SS. (**A**) _H. pylori_ ]99; (**B**) _H. pylori_ G-27; (**C**) _H. pylori_ SS1 and (**D**) _H. pylori_ H707. BB: Brucella broth; FBS: fetal bovine serum; SS: saline solution. Figure 4: _Candida_ growth curves in the presence of BB supplemented with 1%, 5% or 20% FBS or its absence. (**A**) C. _ albicans_ ATCC 90028; (**B**) _C. glabata_ ATCC 90030; (**C**) _C. albicans_ VT-3; (**D**) _C. glabrata_ LEO-37. The highest growth level was achieved using 20% FBS, followed by the cultures with 1% and 5% FBS. In the latter, there was no significant difference identified between these two growth curves \(p>0.05\); however, a highly significant difference was found between the growth curves in SS when compared with the other treatments \(p<0.0001\). Figure 5: **(A)** Wet mount of _H. pylori_ H707–C. _albicans_ VT-3 24 h co-culture in BB-1%FBS showing Y-BLBs (black arrows) and _H. pylori_ cells attached to pseudohyphae (red arrows). (**B**) Wet mount of _C. albicans_ VT-3 cells from a pure culture (negative control) showing the absence of Y-BLBs. Movement of BLBs within yeasts is shown in Video S1. BB-1% FBS: Brucella broth supplemented with 1% of fetal bovine serum. Subsequently, in order to analyze the interaction of the different strains of _Candida_ and _H. pylori_ when co-cultured, the time at which the highest Y-BLBs mean was obtained in each co-culture supplemented with the different FBS concentrations was determined (Figure 7). Once the times with the highest percentages of mean Y-BLBs were obtained, the data were analyzed using [PERSON]'s test to determine if any differences were dependent on the type of bacterial or fungal strains or only on the concentration of FBS. The analysis showed that the most important factor generating the intracellular interaction between _H. pylori_ and _Candida_ was the concentration of FBS in the medium. However, in all the co-culture conditions, higher means were observed between _H. pylori_ strains H707 and J99 with _C. glabrata_ ATCC90030 and LEO-37 strains (Figure 8). The interaction of all co-cultures is shown in Figures S1-S3. Figure 6: Mean percentage of Y-BLBs when _H. pylori_ and _Candida_ spp. were co-cultured in BB supplemented with 1%, 5% or 20% FBS concentrations or in SS. Mean percentages of Y-BLBs remained above 50% in the co-cultures supplemented with 1% FBS. No significant differences were observed between the percentages of Y-BLBs obtained in the co-cultures evaluating the same strains of _H. pylori_ in the presence of different FBS concentrations. Different letters indicate significant differences (_p_ < 0.05). Figure 7: Means of Y-BLBs were obtained for all bacterial strain and yeast strain combinations cultured in BB supplemented with 1%, 5% or 20% FBS or SS during 48 h. This figure shows the means of Y-BLBs obtained when co-culturing _H. pylori_ J99 and _C. glabrata_ ATCC 90030 strains.