https://orcid.org/0000-0002-2944-6001
Figures
Abstract
Background
Helicobacter pylori infections are generally acquired during childhood and affect half of the global population, but its transmission route remains unclear. It is reported that H. pylori can be internalized into Candida, but more evidence is needed for the internalization of H. pylori in human gastrointestinal Candida and vaginal Candida.
Methods
Candida was isolated from vaginal discharge and gastric mucosa biopsies. We PCR-amplified and sequenced H. pylori-specific genes from Candida genomic DNA. Using optical and immunofluorescence microscopy, we identified and observed bacteria-like bodies (BLBs) in Candida isolates and subcultures. Intracellular H. pylori antigen were detected by immunofluorescence using Fluorescein isothiocyanate (FITC)-labeled anti-H. pylori IgG antibodies. Urease activity in H. pylori internalized by Candida was detected by inoculating with urea-based Sabouraud dextrose agar, which changed the agar color from yellow to pink, indicating urease activity.
Results
A total of 59 vaginal Candida and two gastric Candida strains were isolated from vaginal discharge and gastric mucosa. Twenty-three isolates were positive for H. pylori 16S rDNA, 12 were positive for cagA and 21 were positive for ureA. The BLBs could be observed in Candida cells, which were positive for H. pylori 16S rDNA, and were viable determined by the LIVE/DEAD BacLight Bacterial Viability kit. Fluorescein isothiocyanate (FITC)-conjugated antibodies could be reacted specifically with H. pylori antigen inside Candida cells by immunofluorescence. Finally, H. pylori-positive Candida remained positive for H. pylori 16S rDNA even after ten subcultures. Urease activity of H. pylori internalized by Candida was positive.
Citation: Yang T, Li J, Zhang Y, Deng Z, Cui G, Yuan J, et al. (2024) Intracellular presence of Helicobacter pylori antigen and genes within gastric and vaginal Candida. PLoS ONE 19(2): e0298442. https://doi.org/10.1371/journal.pone.0298442
Editor: António Machado, Universidad San Francisco de Quito, ECUADOR
Received: June 19, 2023; Accepted: January 23, 2024; Published: February 8, 2024
Copyright: © 2024 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81860353), the Basic Research Program of Guizhou Science and Technology Plan (No. ZK [2022] 341), the Science and Technology Fund Project of Guizhou Health Commission (No. gzwkj2022-521) and the Scientific Foundation of Guizhou Medical University (No. 20NSP005), and the Foundation of Key Laboratory of Microbiology and Parasitology of Education Department, Guizhou (No. QJJ [2022] 019). These funding obtained from Zhenghong Chen, who had a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: All authors declare no conflicts of interest.
Introduction
Discovered in 1983, Helicobacter pylori (originally Campylobacter pylori) is a spiral-shaped, gram-negative bacterium that often causes acute and chronic gastritis and peptic ulcer disease [1]. It is also closely associated with the development of gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer [1]. In 1994, the International Agency for Research on Cancer of the World Health Organization classified H. pylori as a class I carcinogen [2]. H. pylori infection is a public health problem worldwide [3], and approximately half of the world’s population is infected with it [4]. H. pylori infection is especially higher in less developed countries such as Jordan, where 88.6% of people aged between 15 and 81 years are infected [5]. The infection is generally acquired during childhood and can remain asymptomatic, but H. pylori often exhibits a long-term and chronic infection process [6–8]. The child infection rates are typically around 30%: in Cairo, 33% of children under age of 6 years are infected [9]; in Poland, 32.01% of children between the ages of 6 months and 18 years are infected [10]; and in Changsha, China, 30.6% of infants and toddlers are infected [11].
The main transmission routes for H. pylori are thought to be fecal-oral [12] and oral-oral [13,14], and these routes occur through ingesting contaminated food or water [12,14], communal meals, sharing towels, receiving pre-chewed food from the mother and physical acts such as kissing [15,16]. From this discovery, questions arise about whether close contact, such as communal meals, sharing towels, or kissing can lead to the spread of H. pylori infection [17].
Some studies have suggested that mothers with H. pylori infection may be the main transmission source during childhood [8,18]. A subsequent study confirmed the mother-to-child transmission of H. pylori, a phenomenon that can be attributed to mothers being the primary caregivers and children having more opportunities for oral-oral and fecal-oral transmission [19]. However, H. pylori is extremely fragile and dies quickly after exposure to the air, and in our experiments, we failed to isolate viable isolates from saliva and feces of 50 patients infected with H. pylori (unpublished), most studies reporting on detection of H. pylori in saliva or dental plaque have been performed by polymerase chain reaction (PCR) methods [20–22]. Hamada et al. did not culture H. pylori from the oral cavity or fecal samples, and the ureA gene of H. pylori was positive by PCR amplification [23]. Mao et al. presented a critical discussion of previous studies investigating the potential colonization of the oral cavity by H. Pylori [17]. Therefore, the oral cavity is not a suitable host for H. pylori colonization.
As early as 2013, researchers have proposed that vaginal yeast is the primary reservoir of H. pylori, with the bacteria transmitted from H. pylori-positive mothers to newborns during vaginal delivery or contact with the hospital environment and healthcare workers [24]. However, there has been little focus on investigating alternate transmission routes for H. pylori beyond the commonly recognized oral-oral or fecal-oral routes. Therefore, this study aimed to determine whether H. pylori is present in Candida isolated from vaginal discharge of pregnant women, as well as vaginal discharge and gastric mucosa of patients with digestive diseases.
In this study, Candida was isolated from vaginal discharge in pregnant women and female patients with gastric disease and from gastric mucosa biopsies in the female patients. Using optical and fluorescence microscopy, we identified and observed the bacteria-like bodies (BLBs) in isolated Candida and subcultures. We then PCR-amplified and sequenced H. pylori specific 16S rDNA and cagA genes from Candida cells. Finally, we separately diagnosed H. pylori infection in pregnant women and female patients using the H. pylori antibody test and urea breath test (UBT). The aim of this study was to detect intracellular H. pylori in vaginal and gastric Candida isolates and to explore the potential for transmission of H. pylori from mother to newborn through vaginal Candida during delivery.
Material and methods
Ethics approval and consent to participate
This study was approved by the Human Medical Ethics Committee of Guizhou Medical University in April 2021, following the Ethics Review Document No. 141. All procedures complied with the Declaration of Helsinki. Verbal informed consent was obtained from all participants for their anonymized information to be published in this article.
Samples
This study included 50 pregnant women who underwent prenatal examination in the obstetrics department of Jinyang Affiliated Hospital at Guizhou Medical University from May to October 2021. Samples of vaginal discharge and serum were collected. In addition, 27 gastric biopsies and 22 vaginal discharges were collected from 42 female patients with gastric disease at the Department of Gastroenterology in the People’s Hospital of Qiannan Prefecture (Guizhou Province, China) from May to October 2021. Seven female patients provided samples of both gastric mucosa and vaginal discharge. However, only vaginal discharge samples were obtained from another 15 female patients who did not undergo gastroscopic examination. Finally, 20 individuals were outpatients; therefore, only gastric mucosa samples were collected.
Infection in pregnant women was diagnosed via a quantitative detection of serum H. pylori antibodies. An H. pylori antibody test kit (WAN TAI BRD, Beijing, China) for performing latex immunoturbidimetry (ARCHITECT c16000, Abbott, USA) was used to determine H. pylori IgG. Finally, H. pylori infection in female patients with gastric disease was diagnosed using the 14C-urea breath test (UBT).
Candida isolation and identification
Samples of vaginal discharge and homogenized gastric mucosa were inoculated directly on CHROMagar Candida medium (CHROMagar, Paris, France) following the manufacturer’s protocol and then incubated at 37°C for 24–48 h under aerobic conditions. Candida species were identified based on the color characteristics of different colonies, such as green-blue (C. albicans), metallic blue with pink halo (C. tropicalis), mauve (C. glabrata), pink and fuzzy (C. krusei), and white (other Candida species). Candida was passaged on Sabouraud dextrose agar (SDA, Basebio, Hangzhou, China) with 50 μg/mL chloramphenicol (Solarbio, Beijing, China) medium.
Detection of H. pylori-specific genes in Candida cells
Whole genomic DNA from Candida and control isolates was extracted using the UltraClean ® Microbial DNA Isolation Kit (Qiagen, Germantown, USA). The negative control was C. albicans strain ATCC 10231; the positive control was H. pylori strain 26695, and the blank control was sterile deionised water. H. pylori-specific 16S rDNA, cagA and ureA were PCR-amplified in a 25 μL reaction volume containing 1 μL of forward primer (10 μM, Sangon Biotech Co., Ltd., Shanghai, China), 1 μL of reverse primer (10 μM, Sangon Biotech Co., Ltd.), 2 μL of genomic DNA, 12.5 μL of 2× Taq PCR master mix (Jiangsu CWBiotech Science and Technology Co., Ltd., Jiangsu, China), and sterile deionised water (8.5 μL). Primers’ sequences and thermocycling conditions are described in Table 1.
Amplicons of H. pylori-specific 16S rDNA, cagA and ureA were sequenced at Sangon Biotech and aligned with GenBank sequences using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/).
Alignment and phylogenetic analysis.
The evolutionary history is inferred using the Neighbor-Joining method [27]. Alignment of multiple sequences was carried out using ClustalW with a penalty of 15 for gap opening and 6.66 for gap extension. Based on 1000 replicates, a bootstrap consensus tree was inferred to represent the evolutionary history of the taxa. Collapsed branches correspond to partitions that have been replicated less than 50% of the time in bootstrap replications. Next to each branch are the percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) [28]. Maximum Composite Likelihood was used to determine evolutionary distances and they are expressed in substitutions per site. Using MEGA X, evolutionary analyses were performed on all sequence pairs with ambiguous positions removed (pairwise deletion option).
Subculturing of H. pylori-positive Candida and detection of H. pylori- specific genes from Candida
Helicobacter pylori-positive Candida were passaged on SDA with chloramphenicol 10 times. To eliminate any possible bacterial contamination, yeast isolates were subcultured on SDA with chloramphenicol ten times. Whole genomic DNA was extracted from the 4th and 10th subcultures, and the H. pylori-specific genes were PCR-amplified. Amplicons were sequenced at Sangon Biotech, and genomic alignment was performed using BLAST.
Microscopic observation of BLBs in Candida
Six colonies from the primary culture were randomly selected and placed on slides containing 10 μL of 0.9% saline solution. Coverslips were placed on the samples to search for BLBs under an optical microscope (Nikon, Shanghai, China) equipped with a 100× oil immersion objective lens and camera [29]. Candida albicans strain ATCC10231 was used as a negative control.
Furthermore, Candida cells were stained using the LIVE/DEAD BacLight Bacterial Viability Kit L7012 (Thermo Fisher, Waltham, MA, USA), which uses a mixture of the SYTO 9 green-fluorescent nucleic acid stain and propidium iodide, a red-fluorescent nucleic acid stain. These stains differ in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, the SYTO 9 stain generally labels all bacteria (with intact and damaged membranes) in a population. In contrast, propidium iodide penetrates only bacteria with damaged membranes, revealing the bacterial nature of BLBs and their viability. Briefly, fresh Candida cultures were suspended in a sterile saline solution, and the turbidity was adjusted to 0.5 McFarland standard. A 0.5 mL of each Candida suspension was mixed with 1.5 μL of fluorescent stain containing equal volumes of SYTO 9 and propidium iodide. After a quick vortex and incubation at 25°C in the dark for 15 min, 5 μL of each Candida suspension was placed on a glass slide and examined using the 100× lens of a fluorescent microscope equipped with an integrated camera (Nikon, Shanghai, China).
We selected one H. pylori 16S rDNA positive vaginal Candida strain, one H. pylori 16S rDNA positive gastric Candida strain, and C. albicans ATCC10231 to observe Candida cells using transmission electron microscopy (TEM). The fresh Candida cultures were collected in 1.5 mL Eppendorf tubes (Eppendorf GmbH, Hamburg, Germany), centrifuged at 10,000 × g for 1 min, and washed thrice with 1 mL phosphate-buffered saline (PBS). The precipitate was resuspended in 2.5% (v/v) glutaraldehyde fixative at 4°C for 24 h and subjected to TEM analysis by Shiyanjia Laboratory (http://www.shiyanjia.com).
Detection of intracellular H. pylori antigen by immunofluorescence
This assay was done using Fluorescein isothiocyanate (FITC)-labeled polyclonal anti-H. pylori IgG antibodies at a concentration of 5 mg/mL (Thermo Fisher, Cat # PA1-73161). Each Candida isolate was independently cultured in 10 mL of Brain Heart Infusion (BHI) medium (OXOID, Basingstoke, United Kingdom) at 37°C shaking at 120 r/min for 24 h, then Candida was treated with 32 μg/mL amphotericin B at 37°C shaking at 120 r/min for 24 h [30]. Amphotericin B increases the permeability of yeast cell walls, which then allows antibodies to enter yeast cells. The culture was then centrifuged for 1 min at 10000 r/min and washed three times with 1 mL of PBS (pH 7.2), the precipitation was resuspended in PBS solution and the turbidity was adjusted to 2 McFarland standard. A 100 μL of each Candida suspension was mixed with 1 μL of FITC- labeled anti-H. pylori IgG antibodies, and were incubated for 1 h at 25°C in darkness. A 5 μL volume of each suspension was spotted onto a glass slide and observed by fluorescence microscopy. Fresh cultures of H. pylori 26695 strain and C. albicans ATCC 10231 were used as positive and negative controls, respectively.
Detection of urease activity in H. pylori internalized by Candida
To demonstrate intracellular of H. pylori release from the Candida cells, H. pylori 16S rDNA positive Candida was inoculated into SDA agar medium (pH 7.2) containing 40% urea (Hai bo, Qingdao, China) and 0.01 g/L phenol red at 37°C for 3–5 days under aerobic conditions. In the presence of H. pylori, bacterial urease catalyzes the conversion of urea to ammonia and carbon dioxide, which can be detected by the characteristic red color change in solution [31]. By observing the medium, changing the agar color from yellow to pink is positive for urease and negative for no pink. The C. albicans ATCC 10231 was used as negative controls under aerobic conditions (C. albicans ATCC 10231 isolated from Man with bronchomycosis). The H. pylori 26695 strain was used as a positive control under microaerobic conditions.
Results
Helicobacter pylori infection
Of the 50 pregnant women, 13 tested positive for H. pylori IgG antibodies. Among the 42 female patients, 31 were 14C-urea breath test (UBT)-positive and 11 were UBT-negative. Thus, 13 pregnant women and 31 patients with gastric disease were H. pylori-positive (Fig 1 and S1 File).
N, number of people; H. pylori-Ab (-), H. pylori antibody-negative; H. pylori-Ab (+), H. pylori antibody-positive.
Candida isolation, identification and subcultures
One Candida strain was isolated from each vaginal discharge sample to yield 50 isolates. Based on the color of colonies on CHROMagar Candida medium, we obtained 45 isolates of C. albicans, two isolates of C. glabrata, two isolates of C. tropicalis, and one isolate of the other Candida spp.
Nine vaginal Candida isolates and two gastric Candida isolates were obtained from female patients with gastric disease, including four C. albicans, four C. cruise, one C. glabrata, and two isolates of the other Candida spp. (Fig 1 and Table 2). The Candida colonies were sub-cultured on SDA medium for more than ten generations to remove extracellular bacterial contamination.
Intracellular presence of H. pylori-specific genes within Candida subcultures
Thirteen vaginal Candida isolates from pregnant women, who were positive for H. pylori IgG antibody, were positive for intracellular H. pylori specific 16S rDNA (Figs 2A and S1) and ureA (Fig 2B and S1), and five were also positive for the cagA (Figs 3 and S1). In addition, 13 H. pylori 16S rDNA positive Candida isolates were isolated from pregnant women (Fig 1).
Lane M: Molecular weight marker. Lane B: Blank control (sterile deionized water). Lane C-: Negative control, DNA extracted from C. albicans strain ATCC 10231. Lane C+: Positive control, DNA extracted from H. pylori strain 26695. (a) Lanes 1–5: DNA extracted from Candida harboring BLBs, obtained from vaginal discharge of pregnant women. Lanes 6–8: DNA extracted from Candida harboring BLBs, obtained from vaginal discharge of patients with gastric disease. Lanes 10–11: DNA extracted from Candida harboring BLBs, obtained from gastric mucosa of patients with gastric disease. (b) Lanes 1–11 and Lanes 14–22: DNA extracted from H. pylori 16S rDNA positive Candida. Lanes 12 and 13: DNA extracted from H. pylori 16S rDNA negative Candida (S1 Fig).
Lane M: Molecular weight marker. Lanes 1–2: DNA extracted from Candida isolates in vaginal discharge of pregnant women. Lanes 3–4: DNA extracted from Candida isolates in vaginal discharge of female patients with gastric disease. Lanes 5–6: DNA extracted from Candida isolates in gastric mucosa of patients with gastric disease. Lane B: Blank control (sterile deionized water). Lane C-: Negative control, DNA extracted from C. albicans strain ATCC 10231. Lane C+: Positive control, DNA extracted from H. pylori strain 26695 (S1 Fig).
Eight vaginal Candida and two gastric Candida isolates from female patients with gastric disease, who were positive for UBT, were positive for intracellular H. pylori specific 16S rDNA (Fig 2A), five vaginal Candida and two gastric Candida were also positive for the ureA and cagA (Figs 2B and 3). Only one vaginal Candida isolate from patients who were negative for UBT was negative for H. pylori-specific 16S rDNA and ureA.
These PCR positive Candida isolates were sub-cultured for 10 generations, 16S rDNA and cagA genes of H. pylori were still positive for the tenth subcultures. Sequences of these amplicons showed > 99.8% identity with H. pylori sequences from GenBank. Some sequences of H. pylori 16S rDNA (accession no. ON545841, ON545842 and ON631242) and cagA had been submitted to GenBank (accession no. OM779116, OM779117 and OM812999).
Phylogenetic analyses
The H. pylori 16S rDNA sequences obtained from PCR assays were analyzed to determine the evolutionary history of H. pylori species within Candida cells (Fig 4). Based on BLAST results, the analyses included GenBank reference sequences. These sequences were confirmed to be those of Helicobacter pylori.
This phylogenetic tree was constructed using the GenBank reference sequence (H. pylori SS1) and H. pylori 16S rRNA sequences obtained from Candida. SS1 is a standard strain of Helicobacter pylori. V1-V9 and OR226730 represent sequences of H. pylori 16S rRNA genes from vaginal Candida. ON545841, ON545842 and OQ259931 represent sequences of H. pylori 16S rRNA genes from gastric Candida.
Microscopic observations reveal the presence of live H. pylori in Candida cells
Optical microscopy observations revealed BLBs in 13 of 50 vaginal Candida isolates from pregnant women. We also found BLBs in 10 of 11 Candida isolates from patients with gastric disease, including eight vaginal isolates and two gastric mucosa isolates (Table 2 and Fig 5). No BLBs in C. albicans ATCC 10231. At the same time, BLBs were observed to move rapidly within the Candida vacuole (S1 Video).
Photographs taken at 0, 6, 18, and 38 s (a, b, c and d, respectively) show fast-moving BLBs inside vacuoles. Red arrow indicates the Candida nucleus, and black arrows indicate BLBs. Refer to the attached video of fast-moving BLBs within vacuoles (S1 Video).
Fluorescence microscopy, using the BacLight Bacterial Viability kit, showed the BLBs within Candida cells were viable, as indicated by the green fluorescence (Fig 6). Intracellular H. pylori within Candida was observed by TEM (Fig 7).
The SYTO 9 stain generally labels all bacteria (with intact and damaged membranes) in a population. In contrast, propidium iodide penetrates only bacteria with damaged membranes. (a) C. albicans ATCC10231 strain (negative control). (b) The live and green BLBs are demonstrated in the candida (white arrow), and (c) dead Candida (magnification × 1000).
(a) C. albicans ATCC 10231(absence of high electron density body). (b) High electron density body (white arrow) in vaginal Candida cells. (c) High electron density bodies (white arrow) in gastric Candida cells. Scale bars = 1.0 μm.
Direct immunofluorescence assay confirmed the presence of H. pylori antigen in Candida
The FITC-labeled anti-H. pylori IgG antibodies reacted specifically with intracellular H. pylori within Candida cells as showed by the green fluorescence under fluorescence microscope (Fig 8).
Immunofluorescence micrographs showing: (a) absence of fluorescence in the C. albicans ATCC 10231 (negative control); (b) presence of fluorescence emitted by H. pylori 26695 strain (positive control); (c) presence of fluorescence emitted by H. pylori (white arrows) inside Candida (magnification × 1000).
Detection of urease activity in H. pylori internalized by Candida
To demonstrate of intracellular H. pylori urease activity in Candida, Candida was inoculated with urea-based SDA agar, which changed the agar color from yellow to pink, indicating urease activity. H. pylori ureA positive Candida cells in urea-based SDA agar showed urease activity. C. albicans ATCC 10231 had no urease activity and H. pylori 26695 strain was urease positive.
Consistency between H. pylori infection, BLBs and intracellular H. pylori in Candida
Thirteen Candida isolates were isolated from pregnant women whose H. pylori IgG antibody was positive and H. pylori 16S rDNA was positive in all Candida isolates that harboured BLBs observed by optical microscopy. Interestingly, H. pylori 16S rDNA and BLBs were negative in all vaginal Candida isolates isolated from pregnant women whose H. pylori antibodies were negative (Fig 1). Furthermore, among H. pylori-negative patients, only one vaginal Candida isolate lacked BLBs and was negative for H. pylori 16S rDNA and ureA (Fig 1). These results indicate that H. pylori 16S rDNA was present in Candida isolates with BLBs. Individuals who provided such specimens were therefore infected with H. pylori.
Discussion
Many studies have reported that H. pylori infection exhibits household clustering, with the most likely routes of transmission being oral-oral, fecal-oral, and gastro-oral [8,18,32,33]. High-risk factors for intrafamily transmission include shared towels, mothers feeding pre-chewed food to children, and history of family members with gastrointestinal disease [15]. Despite improvements to social economy, housing, dietary hygiene, and feeding habits within the last 20 years, H. pylori infection remains prevalent in children and even infants. From 2009 to 2011 in China, the overall H. pylori infection rate in newborns was 0.6%, with some variation across major cities, e.g., 0.3% in Beijing, 3.8% in Chengdu [15]. As newborns rarely have the opportunity to eat contaminated foods, this reflects that their low likelihood of being infected with H. pylori through the fecal-oral route. In addition, H. pylori is an oxygen-sensitive bacterium that dies soon after leaving the human stomach [34]. Although in most families, mothers assume the responsibility of taking care of children, children and fathers also have close contact. However, in this study, in nine families where only fathers are positive, children are not infected with H. pylori [35]. Therefore, the infection route to these newborns’ oral cavity is probably directly from their mothers during birth.
Siavoshi et al. reported that vaginal Candida may be a host of H. pylori, sheltering the bacteria even outside the human body [24]. Subsequently, Sanchez-Alonzo et al. showed that 50% of isolated vaginal Candida were positive for H. pylori 16S rDNA, and 32% of such samples were also positive for cagA [29]. CagA is a virulence factor of H. pylori infection and is associated with H. pylori colonization [36]. Tohidpour reported that CagA wass the first bacterial oncolytic protein to rank the H. pylori-mediated adenocarcinoma as the second deadliest cancer type worldwide [37]. These findings corroborate our results. Few reports are available on the relationship between female vaginal Candida and H. pylori or the vertical transmission of H. pylori through the birth canal. Thus, more research is necessary to confirm the intracellular presence of H. pylori in Candida and its potential transmission from mothers to newborns.
We found that 26% of vaginal Candida in pregnant women contained H. pylori-specific nucleic acids, consistent with the H. pylori IgG positive rates (13/50) in pregnant women. Furthermore, H. pylori-specific nucleic acids and BLBs could still be detected even after 10 passages of vaginal Candida colonies. Sequences of H. pylori 16S rDNA alignments showed > 99.8% identity with H. pylori sequences from GenBank. Sequence alignments showed that the base of the H. pylori 16S rDNA and cagA fragments amplified from different Candida isolates had some differences, and no positive amplification from C. albicans control isolates, so sample or laboratory contamination with H. pylori can be excluded.
As with vaginal Candida, H. pylori-specific nucleic acids (16S rDNA and cagA) were present in Candida from the gastric mucosa of H. pylori-positive patients, even after ten passages. Researchers have reported that H. pylori-specific proteins and H. pylori could be detected in gastric yeast by immunofluorescence [38,39]. Amphotericin B increases the permeability of yeast cell walls, which then allows antibodies into yeast cells [30]. In this study, FITC-conjugated anti-H. pylori IgG antibodies could be recruited for localization of H. pylori inside both vaginal Candida and gastric Candida cells treated by amphotericin B using immunofluorescence. Therefore, through Candida, H. pylori could become a facultative intracellular bacterium in the gastrointestinal tract. Sequestration inside yeast vacuoles appears to enhance H. pylori survival in gastric acid [40], and an in vitro study co-incubating H. pylori and C. albicans revealed that H. pylori enters C. albicans vacuoles under unfavorable pH conditions [41]. Thus, H. pylori within Candida may travel from the stomach to the intestinal tract, and then to the vagina, given its closely proximity to the anus [42]. Several studies have confirmed that H. pylori or H. pylori-positive Candida can enter and colonize the vagina [24,43,44], again pointing to the possibility of H. pylori-positive Candida being transmitted to newborns during natural delivery.
We detected urease activity in H. pylori internalized by Candida, which turned the urea-based SDA pink, demonstrating the release of intracellular H. pylori urease from Candida cells, as well as the presence of the H. pylori ureA gene. Heydari et al. reported that Candida may release free H. pylori as a vesicle-encased or free bacterium [45]. While the elimination of numerous extracellular H. pylori isolates can relieve symptoms, Candida reservoirs of H. pylori may cause chronic infections, recurrence, or even carcinogenesis. The fact that H. pylori was found in Candida isolated from the stomach after several passages indicates that H. pylori remains in Candida progeny even after the Candida cells bud.
Additionally, under light microscopy, we observed rapid movement of bacteria within Candida vacuoles, similarly to previous reports on oral Candida [46]. This movement increases the probability that H. pylori can proliferate within Candida cells and use them as a vector for transmission. Although intracellular H. pylori in Candida cannot be isolated and cultured, Heydari et al. reported that the released H. pylori from Candida were detected by immunomagnetic separation [44].
As early as 2014, Siavoshi et al. considered vacuoles of Candida cell as a specialized niche for H. pylori [47]. However, this conclusion has not received much attention, and even was opposed by Alipour and Gaeini [48], who thought that the size of Candida cells was not large enough to accommodate several H. pylori cells. Additionally, the H. pylori isolates cannot be isolated from Candida, which would violate Koch’s postulates [48]. Then, Siavoshi et al. quickly made a reasonable response, insisted on the published research conclusions, and considered that the traditional Koch’s postulates are not applicable to the non-culturable H. pylori in Candida vesicles [49]. The size of a Candida cell is ~3–8 μm [50], the bacterial cell length of H. pylori is ~2–4 μm and its width is ~0.5–1 μm [51]. Although it is difficult for Candida to accommodate several H. pylori cells, under the exposure of antibiotics, H. pylori may lose its cell wall and become a coccoid form, which can be internalized into Candida cells. Therefore, under unfavorable conditions such as exposure to antibiotics and an aerobic environment, the vacuoles of Candida cells provided a special habitat for H. pylori and made it a shelter for H. pylori [39,47]. Candida albicans is an opportunistic fungal pathogen that can colonize host niches at varying pH [52]. In our study, Candida species carrying H. pylori showed detectable urease activity. It suggests a symbiotic relationship between H. pylori and Candida, allowing the yeast to survive in acidic environments.
Overall, our work provides a clue for further in-depth study on the interaction between H. pylori and C. albicans, routes of H. pylori transmission, and pathogenicity.
Conclusion
In conclusion, our results showed that H. pylori and its virulence-related genes were present in Candida from vaginal discharge of pregnant women. We also found H. pylori in vaginal Candida and gastric Candida from female patients with gastric disease, with H. pylori 16S rDNA was still detectable after 10 subcultures of Candida. As a result, Candida may act as a reservoir for H. pylori in women’s gastrointestinal tract, allowing the bacteria to migrate through the intestines to the vagina and eventually infect newborns during delivery.
Acknowledgments
We thank the Department of Obstetrics in Jinyang Affiliated Hospital of Guizhou Medical University and the Department of Gastroenterology in the People’s Hospital of Qiannan Prefecture (Guizhou Province) for collecting specimens used in this study. We also acknowledge our colleagues for their valuable comments on this paper. We would also like to thank Editage [www.editage.cn] for their help with English-language editing.
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