Introduction: The gastrointestinal tract is the main portal of entry into the human body
for food- and water-borne microorganisms. Helicobacter pylori (H. pylori) is
a bacterium involved in peptic diseases with unknown environmental sources
and route of transmission.1
On the one hand, close contact is believed to be the main route of transmission of H. pylori, which occurs from
mother to child and among siblings.2
On the other hand,
H. pylori is considered as a gastric colonizer whose entry
into the human stomach may occur along with the ingestion of food and water.3
However, there is no convincing
evidence to indicate the survival of H. pylori in food 4
and water.5
It has been suggested that food processing
steps exert physical and chemical stresses on H. pylori,
and thus different foods cannot be considered as vehicles
for carrying H. pylori to the human gastrointestinal tract.6
Furthermore, several reports demonstrated that H. pylori
inside foodborne yeast could be protected from stressful
conditions present in different food materials.7
On the other
hand, Staphylococcus spp. are typically found in both
fermented and non-fermented animal and plant foods.8
The coexistence of Staphylococcus and yeast has been
frequently reported, for example, in food and microbial
biofilms.9
A clinically significant yet not fully elucidated
fungal-bacterial interaction is the one occurring between
Candida albicans and Staphylococcus spp.10
Yeasts are ubiquitous unicellular fungi that live as
saprophytes on plant or animal materials, preferentially using sugars as carbon and energy sources.11 Yeasts
are equipped with different hydrolytic enzymes, such
as glycosidases, cellulases, proteinases, and lipases,11-13
which enable them to use different kinds of substrates
and thus thrive in a wide range of environmental niches.
Reports indicate that compared with bacteria, yeasts are
more tolerant of stressful conditions such as acidic pH
and are also able to grow in a wider range of water activity.14
Sugar-rich foods with low water activity are considered
to be stressful materials that are hostile to microbial life,
causing bacterial death due to osmotic shock.15 However,
osmotolerant yeasts not only tolerate the osmotic shock
but are capable of growing under such conditions. It has
been demonstrated that osmotolerant yeasts accumulate
glycerol or other polyols in response to low water activity, maintaining or restoring an inside-directed driving
force for water across their cell membrane.11 Moreover,
these polyols, with their hydroxyl groups, retain intracellular polymers in hydrated form, thus preserving enzyme
activity.16-18 Yeasts that tolerate environments with highsugar and low-water contents are osmotolerant yeasts
encompassing most of the ascomycetes.19 Fresh fruits
with high levels of sugars and other nutrients and
intermediate (15-50%) water content provide favorable
conditions for microbial growth.20 However, bacteria
cannot tolerate the acidic pH of these fruits and are thus
eliminated, allowing osmotolerant yeasts to multiply
and become established as the normal microflora in the
sweet niche of fruits.21 Dried fruits, preserved fruits, and
fruit syrups are also sugar-rich foods with low water
activity that carry osmotolerant yeasts as their normal
microflora.14 Different kinds of sugars that are used as
additives in sweet foods might also carry osmotolerant
yeasts. These yeasts are common contaminants of sugar
factories and those that process concentrated solutions
of sugars.22 Sugars are produced from molasses of sugar
beet or sugar cane that have high microbial contents,
mainly consisting of bacterial spores, yeasts, and molds.
Yeasts and molds do not usually survive the main steps
of sugar manufacturing operations, which involve high
temperatures and reduced water activity. However,
airborne yeasts or those that occur on the surface of
refinery equipment can recontaminate the raw sugar in
the final steps, multiply, and increase their population to
104-106 per gram of sugar.23 Accordingly, most of the
yeast populations in sugar products are postproduction
contaminants.24
In this study, yeast isolates from sugar-rich foods,
fresh fruits, dried fruits, commercial unprocessed and
processed sweet foods, and miscellaneous foods were
examined for the occurrence of intracellular H. pylori
and Staphylococcus spp. by molecular and microscopic
methods. Specific primers were used for the detection
of H. pylori and Staphylococcal 16S rDNA in the total
DNA of yeasts. Light and fluorescence microscopes were
used for observing the live and moving bacteria inside
the yeasts. Moreover, FITC (Fluorescein isothiocyanate)
conjugated antibodies were used for immunodetection of
H. pylori and Staphylococcus spp. inside the vacuole of
yeast cells. The rationale of the study was to demonstrate
that yeasts in popular sugar-rich foods may serve as
reservoirs of H. pylori and Staphylococcus, facilitating
their spread within human populations
Methods: Collection and culture of samples
Sixty samples were collected from high-sugar foods and classified into four groups: fresh fruits (×15): blackberry,
apple, grape, persimmon, peach, fig, banana, white dragon,
red dragon, Saturn peach, strawberry, carrot, plum, and
cantaloupe; dried fruits (×15): whole date, heart of palm,
date cube, raisin, and dried apricot; processed and unprocessed commercial foods (×22): brown sugar, icing
sugar, quince jam, date syrup, sugarcane syrup, sugarcane
foam, cooked beets, kombucha tea, white sugar, sugar
cube, cinnamon-flavored sugar cube, low-calorie sweetener,
Gaz (traditional Persian sweet), rock candy, black grape
syrup, white grape syrup, plain biscuit, and Iranian delight;
miscellaneous foods (×8): pistachio, almond, cashew
nut, walnut, old pickled garlic, and old pickled garlic
syrup. One gram of each food material was inoculated
into 3 mL of brain heart infusion (BHI) broth (Merck,
Germany) and incubated at 30ºC for 7-10 days. A 50-
µL volume of each BHI broth was surface inoculated on
YGC (0.5% yeast extract, 2% glucose, 0.01% chloramphenicol, and 1.5% agar) and observed for the growth of
yeast after 24-48 hours of incubation at 30ºC.
Isolation of yeasts
A single colony was selected from each of the 32
yeast-positive cultures and sub-cultured more than 10
times on YGC agar to ensure the absence of bacterial
contamination. Fresh cultures of yeasts were used for
gram staining and observation of the typical morphology
of yeasts by light microscopy.
PCR-restriction fragment length polymorphism
(RFLP) of the internal transcribed spacer (ITS) region
in 5.8S rDNA
Fresh cultures of yeasts were used for the extraction
of DNA.25 The primer pair used to amplify the ITS region
was ITS-1 (5’-TCCGTAGGTGAACCTGCGG-3’)
and ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’).26
Amplification was performed with yeast DNA as a template
and initial denaturation at 94ºC for 5 min, followed by
35 cycles of denaturation at 94ºC for 1 min, annealing at
55ºC for 1 min, and extension at 72ºC for 2 min, and a
final extension at 72ºC for 10 min. PCR products were
electrophoresed using 1% agarose gel in Tris-borateEDTA (TBE) buffer (0.5x) and digested without further purification, using restriction endonucleases HhaI
(Promega, USA), HaeIII and HinfI (Bioron, Germany).
Restriction fragments were electrophoresed using 2%
agarose, and the size of fragments was determined
according to a 50-1500 bp molecular ladder. Yeasts
were classified into 13 groups according to their RFLP
pattern.26
Amplification and sequencing of 26S rDNA
Amplification of the D1/D2 region of 26S rDNA
of the 32 yeasts was carried out using primers NL1
(5’-GCATATCAATAAGCGGAGGAAAAG-3’) and
NL4 (5’-GGTCCGTGTTTCAAGACGG-3’).27 PCR
was performed with initial denaturation at 94º C for 1
min followed by 35 cycles of denaturation at 94ºC for 1
min, annealing at 50ºC for 1 min, and extension at 72ºC
for 2 min with final extension at 72ºC for 5 min. After
electrophoresis, PCR products of representatives of the
13 RFLP groups with a size of 600 bp were purified,
sequenced, and matched with published sequences in
GenBank by using the BLAST program (https://blast.
ncbi.nlm.nih.gov).
Light and fluorescence microscopy for observation
of intracellular bacteria
Wet mounts were prepared from fresh cultures of the
32 yeasts on YGC agar and examined by light microscopy
to observe the moving bacteria inside the vacuoles of
yeasts. Furthermore, to find out whether bacteria were
alive, a fresh culture of a yeast isolate was used for
staining with the LIVE/DEAD BacLight Bacterial Viability
Kit (L-7012; Molecular Probes, USA) according to the
manufacturer’s instructions. A wet mount was examined
by a fluorescence microscope (Olympus, Tokyo, Japan),
and photographs were taken at different time intervals.
Detection of H. pylori-specific 16S rDNA in yeasts
Total DNA from 32 yeast isolates was examined for
the presence of H. pylori-specific 16S rDNA. PCR was
carried out using primers HP1: 5’-GCAATCAGCGTCAGTAATGTTC-3’ and HP2: 5’-GCTAAGAGATCAGCCTATGTCC-3’.28 A clinical isolate of H. pylori
that was previously identified by amplification and
sequencing of H. pylori-specific 16S rDNA was used as
a positive control. PCR reaction mixture without template
was used as a negative control. PCR was started with
94°C for 3 min and 33 cycles of 94°C for 45 s, 57°C for
184 Osmotolerant Yeasts Carry Intracellular Bacteria
Middle East J Dig Dis/ Vol.12/ No.3/ July 2020
1 min and 72°C for 1 min, followed by 72°C for 5 min.
PCR products were electrophoresed using 1% agarose
gel, and their size was determined using a 50-1500 bp
DNA ladder.
Detection of Staphylococcus-specific 16S rDNA in
yeasts
Amplification of Staphylococcus-specific 16S
rDNA was performed using the primers 16S-F
5-AACTCTGTTATTAGGGAAGAACA-3,29 and 16S-R
5’-CCACCTTCCTCCGGTTTGTCACC-3.30 The PCR
program consisted of an initial denaturation step at 94°C
for 10 min, followed by 35 cycles of denaturation at 94°C
for 45s, annealing at 54°C for 45s and extension at 72°C
for 75s, and a final extension step for 10 min at 72°C.
A clinical isolate of Staphylococcus aureus, which was
previously identified by amplification and sequencing of
Staphylococcus-specific 16S rDNA, was used as a positive control. PCR reaction mixture without template was
used as a negative control. The size of PCR product was determined as above. The PCR product amplified from the
Candida parapsilosis isolate was purified, sequenced, and
matched with published sequences of Staphylococcus
spp. in GenBank. Results of sequence analysis showed
99% similarity to Staphylococcus succinus.
Localization of H. pylori and Staphylococcus spp.
inside the yeast vacuole using direct immunofluorescence
assay
Localization of H. pylori and Staphylococcus spp.
inside the vacuole of C. parapsilosis was performed by
direct immunofluorescence (IF) assay. FITC-conjugated
IgY-HP prepared against H. pylori in hen,31 and FITCconjugated IgG-ST prepared against Staphylococcus
spp. in rabbit,32 were used for detection of H. pylori and
Staphylococcus spp. inside the yeast’s vacuole. IF assay was
performed according to Hašek.33 A fresh culture of yeast in
YG (0.5% yeast extract and 2% glucose) broth, was fixed
with 7.4% paraformaldehyde for 120 min while shaking.
After washing with 0.1 M potassium phosphate citrate
buffer (KCP), fixed cells were permeabilized using
lyticase (L4025; Sigma) and Triton X-100. Cells were
washed and resuspended in 0.4 M PIPES buffer containing
FITC-labeled antibodies and 0.01% evans blue solution
(for color contrast) and incubated at room temperature
for 60 min. After washing three times with PIPES, a
10-µL volume of yeast suspension was smeared onto a
glass slide, air-dried, covered with mounting oil (Invitrogen, USA) and examined by fluorescence microscopy.
Fresh cultures of H. pylori and Staphylococcus aureus
were used as positive controls. A negative control yeast
(a yeast with no amplification of H. pylori and Staphylococcus 16S rDNA) was used to demonstrate the lack of
non-specific interaction of antibodies.
Results: Isolation of yeasts
Microscopic examination of gram-stained smears
of yeast colonies on YGC agar showed typical yeast
morphology. Of 60 samples, 32 (53.3%) were positive
for yeast growth. Yeast-positive samples included 10 of
15 (66.6%) fresh fruits, 8 of 15 (53.3%) dried fruits, 11
of 22 (50%) commercial foods, and 3 out of 8 (37.5%)
miscellaneous foods.
Molecular identification of yeasts
Amplification of the ITS region of 5.8S rDNA from the
32 yeasts revealed bands with a size of 370 to 880 bp, which
were digested with restriction endonucleases. Yeasts were
classified into 13 groups according to their PCR-RFLP
pattern. RFLP group 12 with five isolates of C. albicans,
group 8 with four isolates of Meyerozyma guilliermondii,
group 5 with four isolates of Candida diversa, and group
1 with four isolates of Pichia kudriavzevii contained the
highest number of yeast isolates. In the fresh fruits group,
isolated yeasts included Zygosaccharomyces bailii (×2), P.
kudriavzevii (×2), Pichia pastoris (×1), Zygosaccharomyces
mellis (×1), Metschnikowia pulcherrima (×1), C. diversa
(×1), and Candida catenulata (×2) (table 1).
In the dried fruits group, isolated yeasts included P.
kudriavzevii (×1), Meyerozyma guilliermondii (×3), Saccharomyces cerevisiae (×1), C. parapsilosis (×2), and
Wickerhamomyces anomalus (×1) (table 2). In the commercial foods group, isolated yeasts included C. albicans (×3), Meyerozyma guilliermondii (×1), P. kudriavzevii (×1), Saccharomyces cerevisiae (×2), C. diversa (×2),
C. parapsilosis (×1), and Yarrowia lipolytica (×1) (table
3). In the miscellaneous foods, isolated yeasts included C.
albicans (×2) and C. diversa (×1) (table 4).
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186 Osmotolerant Yeasts Carry Intracellular Bacteria
Table 1: Frequency of Helicobacter pylori 16s rDNA and Staphylococcus 16s rDNA in 10 yeast isolates from fresh fruits
Samples RFLP group Sequencing result
(26S rDNA)
H. pylori16S rDNA
Staphylococci16S rDNA
Blackberry 2 1 P. kudriavzevii - -
Strawberry 2 Z. bailii + -
Grape 3 Z. mellis - -
Persimmon 4 M. pulcherima - -
Peach 2 Z. bailii + -
Fig 5 C. diversa + -
Banana 6 P. pastoris + -
White dragon 7 C. catenulata + -
Red dragon 7 C. catenulata + -
Carrot 1 P. kudriavzevii + -
Table 2: Frequency of Helicobacter pylori 16s rDNA and Staphylococcus 16s rDNA in eight yeast isolates from dried fruits
Samples RFLP group Sequencing result
(26S rDNA)
H. pylori16S rDNA
Staphylococci16S rDNA
Date 2 1 P. kudriavzevii - -
Date 3 8 M. guilliermondii - -
Date 4 9 S. cerevisiae + +
Date 5 8 M. guilliermondii + +
Date 6 10 W. anomalus + +
Date 7 11 C. parapsilosis + +
Date 8 11 C. parapsilosis + +
Heart of palm 8 M. guilliermondii + -
Table 3: Frequency of Helicobacter pylori 16s rDNA and Staphylococcus 16s rDNA in 11 yeast isolates from processed and unprocessed
commercial foods
Samples RFLP group Sequencing result
(26S rDNA)
H. pylori16S rDNA
Staphylococci16S rDNA
Brown sugar 1 12 C. albicans + -
Brown sugar 2 12 C. albicans + -
Icing sugar 8 M. guilliermondii - +
Quince jam 1 P. kudriavzevii + -
Date syrup 13 Y. lipolytica + -
Sugarcane syrup 12 C. albicans + -
Sugarcane foam 9 S. cerevisiae + -
Cooked beet 1 5 C. diversa - +
Cooked beet 2 11 C. parapsilosis + -
Cooked beet 3 5 C. diversa + -
Kombucha tea 9 S. cerevisiae + +
Table 4: Frequency of Helicobacter pylori 16s rDNA and Staphylococcus 16s rDNA in three yeast isolates from miscellaneous samples
Samples RFLP group Sequencing result
(26S rDNA)
H. pylori16S rDNA
Staphylococci16S rDNA
Pistachio 5 C. diversa + -
Old pickled garlic 12 C. albicans - -
Old pickled garlic syrup 12 C. albicans - -
Middle East J Dig Dis/ Vol.12/ No.3/ July 2020
Light and fluorescence microscopy of yeast
Light microscopic examination of wet mounts prepared from cultures of the 32 isolated yeasts showed the
occurrence of bacteria inside the vacuole of all the yeast
cells (Fig.1 A). Live/Dead staining of yeast cells confirmed the viability of intracellular bacteria (Fig.1 B).
Photographs taken from a stained wet mount of yeast,
at three-time intervals, showed live and moving bacteria
inside the yeast cell vacuole (Fig.1 C1-C3).
Detection of H. pylori- and Staphylococcus-specific
16S rDNA in yeasts
The amplified product of H. pylori-specific 16S rDNA
with a size of 521 bp was detected in 23 of 32 (71.8%)
yeast isolates. The frequency of H. pylori-positive yeasts
in different groups of samples was determined to be 70%
(7out of 10) in the fresh fruits group: Z. bailii (×2), C.
diversa (×1), P. pastoris (×1), C. catenulate (×2), and
P. kudriavzevii (×1) (table 1); 75% (6 out of 8) in the
dried fruits group: Meyerozyma guilliermondii (×2), C.
parapsilosis (×2), W. anomalus (×1) and Saccharomyces cerevisiae (×1) (table 2); 81.8% (9 out of 11) in the
commercial foods group: C. albicans (×3), P. kudriavzevii
(×1), Y. lipolytica (×1), Saccharomyces cerevisiae (×2),
C. diversa (×1), and C. parapsilosis (×1) (table 3); and
33.3% (1 out of 3) in miscellaneous foods: C. diversa
(×1) (table 4). Staphylococcus-specific 16S rDNA with
a size of 750 bp was detected in 22.2% (8 out of 36)
of yeast isolates. The frequency of Staphylococcus spp.-
positive yeasts was determined to be 62.5% (5 out of
8) in the dried fruits group (Saccharomyces cerevisiae,
Meyerozyma guilliermondii, W. anomalus, and 2× C.
parapsilosis) and 27% (3 out of 11) in the commercial
foods group (Saccharomyces cerevisiae, Meyerozyma
guilliermondii and C. diversa) (tables 2 and 3). Yeast isolates from fresh fruits, and miscellaneous foods did not
carry Staphylococcus-specific 16S rDNA (tables 1 and
4). The frequency of yeasts containing both H. pylori and
Siavoshi et al. 187
Fig.1: Light and fluorescence microscopy of yeast. A) Light microscopy of yeast cells shows intracellular bacteria (IB)
inside yeast’s vacuole (V). B) Live intracellular bacteria (IB) appeared as green spots in the vacuole (V) of stained
yeast cells. C1-C3) Photographs taken at three-time intervals (0, 5, and 10 seconds) show the moving bacteria. Original
magnification x 1000.
Middle East J Dig Dis/ Vol.12/ No.3/ July 2020
Staphylococcus spp. was 62.5% in dried fruits and 9%
in commercial foods. Altogether, among the 32 isolated
yeasts, 17 (53%) were H. pylori-positive only, two (6%)
were Staphylococcus spp.-positive only, six (18.7%)
were positive for both H. pylori and Staphylococcus
spp., and seven (21.8%) were negative for both.
Localization of H. pylori and Staphylococcus spp.
inside the yeast vacuole using direct immunofluorescence
assay
Specific interaction of FITC-IgY-HP (Fig.2, B and
C) or FITC-IgG-ST (Fig.2, F and G) with intracellular
bacteria confirmed the identity of bacteria as H. pylori
or Staphylococcus spp. and their localization inside
yeast’s vacuole. Dark vacuole of negative control yeast
without fluorescent spots indicated the lack of non-specific
interaction of antibodies (Fig.2, D and H)
Conclusion: Carbohydrates are the most popular foods consumed by
humans worldwide. In addition to be a major carbon source
for building living cells, their sweet taste, and energy sion afford these compounds a very special place in the human
diet. Sweet foods are recognized as comfort foods because
their consumption leads to elevation of serotonin, the known
anti-depressant neurotransmitter that reduces pain and
regulates sleep and the biological clock.34 Sugars are also
used to improve the quality of foods due to their functional
properties.35 Furthermore, sugar and salt are the oldest
preservatives that, when added to food materials, protect
them against microbial spoilage by producing high osmotic
pressure. Accordingly, sugars are frequently added to a
variety of foods and beverages. Bacteria cannot withstand
the osmotic stress of sugar-rich environments and die,
while osmotolerant yeasts survive and even multiply.36
Fresh and dried fruits, fruit juice, and refined sugar
products are sugar-rich environments of plant origin that
cause stress to microbial cells by reducing water activity,
changing cell turgor pressure, and destabilizing macromolecules.15 This might indicate that floral nectar and
fruits have evolved to accumulate high concentrations of
sugar to protect the fertile parts of plants from microbial
attack.37,38 However, osmotolerant yeasts that show maximum fitness in high concentrations of sugars,39 increase
their population and become established as the normal
microflora of such sugar-rich plant environments, including
floral nectar and fruits.40 These symbiotic yeasts, while
feeding on plants, stimulate plant metabolism, and inhibit
phytopathogens.41,42 Insects, which play an important
role in pollination and reproduction of plants, feed on
yeasts and carry them from the soil to plants and disperse
them within plants during pollination.43 It appears that
symbiosis of yeasts with insects and plants is an important
and inevitable evolutionary event.44 Accordingly, yeasts
as permanent associates of plants, enter the human digestive
system through the consumption of sugar-rich and plantderived food products.
Yeasts with high potential for genotypic 45,46 and
phenotypic 47 plasticity are permanent symbiotic inhabitants of plants,41,42 insects,48 animals,49 and humans 50 in
a wide range of environments.37,51 Accordingly, it is not
surprising that fungi with these sophisticated properties
have evolved to serve as a unique niche for sheltering
the endosymbiotic bacteria.52 In our previous studies,
H. pylori-specific genes were detected in oral,53 gastric,54 vaginal,55 and foodborne 7 yeasts. Furthermore, H.
pylori-specific proteins were detected in the protein pool
of gastric yeasts by western blot technique,56 and intracellular H. pylori was localized in the vacuole of Candida yeast by FITC-IgY-HP.31 Results of similar studies
performed in our lab showed the detection of Staphylococcus- specific genes 57 and proteins 32 in gastric yeasts as
well as staphylococcal localization inside the vacuole of
gastric yeast by immunodetection and FISH methods.32 It
was proposed that inside the vacuole of Candida yeast,
H. pylori, or Staphylococcus are protected from environmental stresses and provided with nutrients for survival
and multiplication. Accordingly, yeast was suggested as a
potent reservoir of H. pylori and Staphylococcus.
7,53,54,58
Among the 32 yeasts isolated in this study, 17 (53%)
were H. pylori-positive only, two (6%) were Staphylococcus spp.-positive only, six (18.7%) were positive
for both H. pylori and Staphylococcus spp., and seven
(21.8%) were negative for both bacteria. Among the sugarrich foods studied, dates showed the greatest potential for
supporting intracellular H. pylori (75%) and Staphylococcus spp. (62.5%), or both (62.5%) in yeasts.
The frequency of H. pylori-positive yeasts in fresh
fruits, dried fruits, and commercial foods was 70–81.8%.
Fresh fruits contain high levels of sugars, other nutrients, and
intermediate water activity that favor microbial growth.
However, acidic pH eliminates bacteria and provides
appropriate conditions for fungal growth. The natural
microbiota of fruits is commonly composed of yeasts such as
Candida, Pichia, Saccharomyces, Hanseniaspora, and
Zygosaccharomyces.21 Dried fruits such as date fruits, in
addition to sugar, contain salts and minerals, fatty acids,
amino acids, proteins, and vitamins, including B1, B2,
and B3. Furthermore, dates are rich in different kinds
of sterols 59,60 that are precursors of ergosterol involved
in the synthesis of yeast membranes.61 It is noteworthy that sterols also serve as precursors of cholesterol,
an important constituent of H. pylori cell membrane.62
Commercial foods such as sugar cubes, granulated white
and brown sugars, and other related high-sugar products
are frequently used as sweet additives to tea, coffee, and
sherbets or to formulated foods such as desserts and pastries.
These compounds with high sugar content could carry
yeasts either of plant origin or introduced as post-operation
contaminants.63
Among the miscellaneous foods investigated, the two
C. albicans isolates from old pickled garlic, and old pickled
garlic syrup did not contain H. pylori or Staphylococcus
spp. However, the three C. albicans isolates from commercial foods, and eight out of nine Candida spp. from
other foods carried H. pylori. This might indicate that
long storage of pickled garlic in vinegar, although favoring
the survival of C. albicans yeasts, could exert a negative
effect on the survival of intracellular bacteria, leading to
a reduction in bacterial copy number such that bacterial
DNA was not detectable by PCR. Negative PCR results
have been suggested to result from failure in the detection
of bacterial genes due to low bacterial copy number,
inadequate amount of extracted DNA,64 or lack of primer
recognition sites in bacterial DNA due to variation in the
target sequence.65
Results of this study showed that sugar-rich foods,
whether naturally sweet or containing added sugar, are
carriers of osmotolerant yeasts that could contain H. pylori
Siavoshi et al. 189
Middle East J Dig Dis/ Vol.12/ No.3/ July 2020
and/or Staphylococcus spp. Detection of H. pylori- and/
or Staphylococcus-specific genes shows the probable
occurrence of multiple endosymbiotic bacteria in the
vacuoles of yeasts with different frequencies. Microscopic
observations of bacteria in new generations of yeasts
along with amplification of H. pylori- and Staphylococcusspecific genes from consecutive generations indicate that
new yeast cells can inherit the intracellular bacteria as
part of their vacuolar content. Extensive studies on the
intracellular existence of non-culturable bacteria inside
arbuscular mycorrhizal fungi indicated that the fungal
vacuole provided a nourishing and protective niche for
the endosymbiotic bacterium ‘Candidatus Glomeribacter
gigasporarum’ (CaGg), facilitating its replication and
transmission to the next generation.52 Moreover, the
occurrence of two types of endosymbiotic bacteria has
been reported in arbuscular mycorrhizal fungi: the gramnegative beta proteobacterium CaGg, and a gram-positive
molicutes-related endobacterium.
66 It is noteworthy that
microscopic observation of bacterial structures inside
the vacuoles of yeast isolates with negative results for
amplification of H. pylori or Staphylococcus genes suggests
the likelihood of the occurrence of other intracellular
bacteria yet to be identified.
Yeasts enter the food cycle of animals, including humans,
through the consumption of sugar-rich and plant-derived
foods. Yeasts are able to survive in the human gastrointestinal tract due to their high potential to adapt to
different stressful conditions and return to natural soils
when excreted. It can be concluded that different yeasts
that occur in the soil are more or less representatives of
the yeast populations of plant and animal life above the
soil surface. In this fashion, yeasts establish more or less
similar populations in soils, plants, and animals, including
humans. In all the steps of the food cycle, yeast may
carry its intracellular bacteria and spread it to different
hosts such as insects, plants, and animals. Accordingly,
the intracellular occurrence of H. pylori, Staphylococcus
spp. and probably other bacteria inside the yeast could be
regarded as a sophisticated survival strategy of bacteria
that evolved along the evolutionary path. Overall, yeasts
may be regarded as permanent reservoirs of bacteria, and
thus bacteria will exist as long as yeasts persist