All protocols were approved by the Human Medical Ethics Committee of Guizhou Medical University (ethics review approval number: 2022-40). All procedures, including the protocol for obtaining informed consent, were performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all participants or their legal guardians prior to study initiation.

The H. pylori strain ATCC 700392 (commonly referred to as H. pylori 26695) was used. For Candida, we used C. albicans ATCC 10231 (Ca10231) as well as clinical Candida isolates V51 and J115 isolated from vaginal secretions, W49 isolated from gastric biopsy specimens, and F49 from fecal samples. All strains were maintained at the Guizhou Key Laboratory of Microbiology and Infectious Disease Prevention and Control.

Fecal samples were collected from 399 patients with gastrointestinal disorders at the Department of Gastroenterology of the Affiliated Cancer Hospital, Guizhou Medical University. To diagnose H. pylori infection, the colloidal gold method was employed for the detection of H. pylori antigen in stool (S_HpAg) (Gisbert et al., 2006). The presence of H. pylori antigen in the feces was detected using a Wondfo H. pylori Antigen Detection Kit (Guangdong, Wondfo). This kit employs the colloidal gold method to detect H. pylori antigen in feces, and is a common method for diagnosing H. pylori infection. Fecal samples were used for the isolation and culture of Candida. A small quantity of fecal sample was taken and added to sterile physiological saline to prepare a suspension. Subsequently, 0.1 mL of the sample should be added to Sabouraud dextrose agar (SDA, Basebio, Hangzhou, China) containing 50 μg/mL chloramphenicol for the isolation and cultivation of Candida. Candida was inoculated onto CHROMagar Candida medium (CHROMagar, Paris, France) at 37°C for 24–48 for 24 France at 37 e) at 37 Candida species were identified based on colony color characteristics: green-blue (C. albicans), metallic blue with a pink halo (C. tropicalis), mauve (C. glabrata), pink and fuzzy (C. krusei), and white (other Candida species). The isolated Candida was stored in 20% glycerol solution at -80°C.

The H. pylori 26695 strain was cultured in brain heart infusion (BHI) medium (OXOID, Basingstoke, United Kingdom) supplemented with 7% defibrinated sheep blood (Biological Industries, Henan, China) and incubated under microaerobic conditions at 37°C for 48–72 h. Ca10231 and Candida isolates were cultured on SDA supplemented with 50 μg/mL chloramphenicol (Solarbio, Beijing, China). Candida cultures were incubated aerobically at 37°C for 24 h.

The H. pylori 26695 strain and Ca10231 strains were cultured separately under specific microaerobic and aerobic conditions, respectively, at 37°C. Prepare suspensions of the H. pylori 26695 strain and Ca10231 strain separately using sterile physiological saline at concentrations corresponding to 5.0 McFarland turbidity and 0.5 McFarland turbidity, respectively. Take 1 mL of H. pylori 26695 bacterial suspension and 0.1 mL of Ca10231 suspension, respectively, and add them to 10 mL of BHI liquid medium containing 10% fetal bovine serum. Cultivate under microaerobic conditions at 37 medium 120 r/min for 24 h. Take 100 μL of the post-culture suspension and inoculate it onto SDA medium supplemented with 50 μg/mL chloramphenicol for subsequent cultivation.

Candida cells in each experimental group were sub-cultured up to the fifth generation in SDA supplemented with 50 μg/mL chloramphenicol. Subsequently, a specific H. pylori gene within each generation of Candida was examined following a previously established protocol (Šeligová et al., 2020). DNA used as controls in the polymerase chain reaction (PCR) amplification process was extracted from pure cultures of H. pylori 26695 (positive control) and Ca10231 (negative control). Briefly, Candida cells were suspended in distilled water, and the DAAN nucleic acid extraction or purification reagent (magnetic bead method) was used for DNA extraction from the samples. PCR assays were performed using primers targeting H. pylori 16S rDNA to identify H. pylori among Candida. The primer sequences used for PCR are listed in Table 1.

16S rDNA amplification was performed in a thermal cycler for the first reaction with 94°C for 3 min, 37 cycles of the external amplification reaction (94°C, 45 s; 55°C, 1 min; 72°C, 1 min), 72°C for 5 min, and a hold at 14°C. Use the first-round amplification product as the template for the second round of amplification. The temperature cycle for the second reaction was the same; however, the number of cycles was reduced to 25.

To avoid contamination, negative and blank controls were introduced after every sample in the experiments.

The CacoHp strains were continuously passaged up to two times. 1 × 10⁶ GES-1 cells were grafted into a 6-well plate and cultured in serum-free RPMI 1640 medium for 12 h. After the cells adhered, 1 × 10⁵ CacoHp and Ca10231 cells were added. After 24 h of culture, the Candida cells adhering to GES-1 cells were counted.

The CacoHp strains were continuously passaged up to five times. A total of 1 × 10⁶ CacoHp and Ca10231 strains were inoculated into the YPD liquid medium and incubated for 24 h under microaerobic conditions. Following incubation, the cultures of CacoHp and Ca10231 were centrifuged and filtered through a 0.22-μm bacterial filter to obtain sterile culture filtrates. These filtrates were further diluted fivefold with RPMI 1640 medium and co-cultured with GES-1 cells for 24, 48, and 72 h. The cytotoxic effects of the filtrates on GES-1 cells were assessed using a Cell Counting Kit-8 (CCK-8). The cell inhibition rate was calculated as follows: Inhibition Rate (%) = [(Absorbance of Control Group–Absorbance of Experimental Group)/Absorbance of Control Group] × 100%.

In this study, urea-SDA medium was used to detect the urease activity of Candida. Phenol red was added to the SDA medium as an indicator, and urea was added as a substrate for the urease reaction. Under slightly alkaline conditions, phenol red turns red, causing the medium to change color from yellow to red. Urea and phenol red were added to the SDA medium to prepare a urea SDA medium containing 5% urea and 0.01% phenol red. Candida were inoculated in freshly prepared urea SDA medium and incubated under aerobic conditions at 37 °C for 1–3 days. H. pylori 26695 was inoculated onto a urea Columbia serum agar plate (CSA) (5% urea, 10% serum) under microaerobic conditions as a positive control.

Candida strains cultured for 24 h in SDA supplemented with chloramphenicol were removed and washed twice with Phosphate-buffered saline (PBS) (pH 7.4) to ensure cleanliness. Subsequently, the fixed fluid diethyl pyrocarbonate (DEPC) -treated water was immediately added, followed by incubation for a minimum of 12 h. Candida samples were dehydrated using a gradient alcohol-paraffin embedding method. A DEPC dilution wash was performed afterwards. Depending on the tissue fixation time, the slices were boiled in a retrieval solution for approximately 10–15 min and allowed to cool naturally. Objective Candida cells were marked using a liquid blocker pen based on their characteristics. Proteinase K working solution (20 μg/mL) was applied to cover the objectives and incubated at 37 °C for 15 min. Washing was performed using pure water, followed by three washes with PBS (pH 7.4), each lasting 5 min. Finally, every section was incubated with a pre-hybridization solution at 37 °C for 1 h.

The pre-hybridization solution was removed and replaced with a hybridization solution containing 1 μM of the fungal 18S rRNA probe (5’-CY3-GTGACAAGCATATGACTAC-CY3-3’). The sections were incubated in a humidity chamber at 40°C for overnight hybridization. The hybridization solution was then removed, and the sections were washed in 2 × saline sodium citrate (SSC) for 10 min at 37°C, followed by two washes in 1 × SSC for 5 min each at 37°C, and a final wash in 0.5 × SSC for 10 min at room temperature. Subsequently, the pre-hybridization solution was removed again, and a hybridization solution containing 1 μM of H. pylori 16S-1 (5’-FAM-GGAGTATCTGGTATTAATCATCG-FAM-3’) probes was added. The sections were incubated in a humidity chamber and hybridized overnight at 40°C. The hybridization solution was then removed, and the sections were washed in 2 × SSC for 10 min at 37°C, followed by two washes in 1 × SSC for 5 min each at the same temperature, and a final wash in 0.5 × SSC for 10 min at 25°C.

For nuclear counterstaining, the sections were incubated with Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) in the dark for 8 min before mounting. Microscopic examination and imaging were conducted using a fluorescence microscope. DAPI emitted blue fluorescence under ultraviolet (UV) excitation between 330 and 380 nm, with an emission wavelength of 420 nm; FAM emitted green fluorescence under excitation between 465 and 495 nm and an emission wavelength of 515–555 nm; and CY3 emitted red fluorescence upon excitation between 510 and 560 nm, with an emission wavelength of approximately 590 nm.

Candida strains isolated from feces, vaginal secretions, and gastric mucosa were cultured on SDA medium for five generations before RNA extraction using the phenol-chloroform method. ABScript III RT Master Mix with gDNA Remover was used to generate complementary DNA (cDNA) from 1 μL of total RNA for qPCR analysis with ChamQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China) on a CFX96 Real-time system (Bio-Rad, Hercules, CA, United States). The primer sequences used for qPCR are listed in Table 2 (Raghwan and Chowdhury, 2014).

qPCR Testing process was performed in CFX96 Real-time system with 95°C for 30 s, 40 cycles of the external amplification reaction [95°C, 10 s; 60°C, 30 s (Collect fluorescence signals)]. The test results are described qualitatively.

Candida was cultured in the BHI medium (OXOID) at 37°C with shaking at 120 r/min for 24 h. The Candida bacterial fluid was centrifuged and washed thrice with saline to obtain a Candida bacterial suspension with a McFarland standard concentration of 2. Fluorescein isothiocyanate (FITC)-labeled anti-H. pylori IgG antibodies (Cat. No. PA1-73161; Thermo Fisher Scientific) were reconstituted at a dilution of 1:100 to ensure thorough mixing, and the mixture was incubated in a light-protected environment at 25°C for 1 h. A total of 10 μL of the suspension was applied to a glass microscope slide, covered with a coverslip, and examined under a fluorescence microscope at 1,000 × magnification. Ca10231 was used as a negative control.

Candida isolates cultured on SDA supplemented with chloramphenicol for 24 h were used to prepare Candida suspensions with a McFarland standard concentration of 2. A total of 2 μL of LIVE/DEAD^® BacLight™ Bacterial Viability Kits (Thermo Fisher Scientific) was added to 1 mL of Candida bacterial suspension and incubated for 15 min at 25°C away from light. A total of 10 μL of the suspension was applied to a glass microscope slide, covered with a coverslip, and examined under a fluorescence microscope at 1,000 × magnification. Ca10231 was used as a negative control.

Data analysis was conducted using GraphPad Prism 9.5.0 software. Measured data were presented as the mean ± standard deviation, and one-way analysis of variance (ANOVA) was employed for comparisons between two groups. Correlation analysis was conducted using the Point-Biserial correlation coefficient. When P < 0.05, the difference was deemed statistically significant.

The FISH and PCR results indicated the presence of H. pylori nucleic acids in H. pylori 16S rDNA-positive Candida. To further confirm the presence of H. pylori in Candida, we employed a direct immunofluorescence assay to detect H. pylori antigens within Candida cells. Under a fluorescence microscope, no green fluorescence was observed in the Ca10231 strain. However, H. pylori 16S rDNA-positive Candida exhibited distinct green fluorescence, indicating specific binding of FITC-labeled anti-H. pylori IgG antibodies with H. pylori antigens inside Candida cells (Figure 4). Therefore, the presence of H. pylori antigens was confirmed in H. pylori 16S rDNA-positive Candida cells.

The LIVE/DEAD^® BacLight Bacterial Viability Kit was used to assess bacterial cell membrane integrity. Bacteria with intact membranes fluoresced green, indicating metabolic activity, while those with compromised membranes, suggestive of cell death, fluoresced red. Green fluorescent signals were observed within H. pylori 16S rDNA-positive Candida, indicating the presence of H. pylori with intact membranes and the potential viability of H. pylori within Candida (Figure 5).