The intestinal mycobiota and its relationship with overweight, obesity and nutritional aspects

Abstract

Background:
The fungal community of the gastrointestinal tract has recently become of interest, and knowledge of its relationship with the development of obesity is scarce. The present study aimed to evaluate the cultivable fungal fraction from the microbiota and to analyze its relationship with obesity.

Methods:
Samples were taken from 99 participants with normal weight, overweight and obesity (n = 31, 34 and 34, respectively) and were cultivated in selective medium, and the cultivable yeasts were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anthropometric and biochemical measures were also evaluated.

Results:
Eutrophic, overweight and obese groups presented concentrations of 1.6, 2.16 and 2.19 log10 colony-forming units g-1 yeast, respectively. Ascomycota and Basidiomycota were the two identified phyla. At the genus level, Candida spp. showed a relatively high prevalence, and 10 different species were detected: Candida glabrata, Candida orthopsilosis, Candida lambica, Candida kefyr, Candida albicans, Candida krusei, Candida valida, Candida parapsilosis, Candida utilis and Candida humilis (with relative abundances of 71.72%, 5.05%, 21.21%, 6.06%, 29.29%, 27.27%, 8.08%, 16.16%, 1.01% and 2.02%, respectively).

Conclusions:
The obese group presented a higher prevalence of Candida albicans. Furthermore, Candida albicans, Candida kefyr and Rhodotorula mucilaginosa showed a high positive correlation with obesity, weight gain and fat mass and showed a negative correlation with high-density lipoprotein and lean mass, parameters related to weight loss.

Full text

J Hum Nutr Diet. 2021;00 :1–11. wileyonlinelibrary.com/journal/jhn
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© 2021 The Brit ish Dietetic Associat ion Ltd.
INTRODUCTION
Obesity is a worldwide epidemiologic pathology charac-
terized by fat mass storage in the body, largely abdominal
cavity fat.1 Obesity is a multifactorial pathology defined as
the result of an imbalance between energy intake and energy
expenditure.2,3 It has been reported that the gut microbiota
is an important factor involved in obesity development.1 The
human gastrointestinal tract harbors a complex commu-
nity of microorganisms called the intestinal microbiota. Of
these microorganisms, the main actors are bacteria, mainly
Lactobacillus spp. and Bifidobacterium spp.4 The intestinal
microbiota is considered as a metabolic organ that forms
part of human physiology.5 It is well documented that this
organ possesses important functions in the body, includ-
ing food digestion6 immune system modulation, metabolic
RESEARC H PA PER
The intestinal mycobiota and its relationship with overweight,
obesity and nutritional aspects
RicardoGarcía-Gamboa1 | Manuel R.Kirchmayr1 | Misael SebastianGradilla-Hernández2 |
VicentePérez-Brocal3,4 | AndrésMoya3,4,5 | MariselaGonzález-Avila1
Received: 15 October 2020
|
Accepted: 5 Janua ry 2021
DOI : 10.1111/j hn.12 864
1Centro de Investiga ción y Asistencia en
Tecnología y Dis eño del Estado de Jalisco,
Guada lajara, Mexico
2Tecnologico de Monte rrey, Escuela de
Ingenieria y Cienci as, Zapopan, Ja lisco,
México
3Fundación para el Fomento de la
Investigación Sa nitaria y Bioméd ica de la
Comunit at Valenciana (FISABIO), València,
Spain
4Consorcio de Invest igación Biomédica
en Red de Epidemiologí a y Salud Pública
(CIBERE SP), Madrid , Spain
5Integrative Systems Biology Institute
(I2SysBio) Universitat de València and
Consejo Superior de I nvestigaciones
Científ icas (CSIC), València, Spa in
Correspondence
Maris ela González-Avila, Ex vivo Dige stion
Laboratory, CIATEJ, Nor malistas No.800, col
Colinas de la Normal, C.P. 44270, Guadalajara
Jalisco, Mexico.
Email: mgavila@ciatej.mx
Fundin g information
CONACYT, Grant/Award Number: 662 891
Abstract
Background: The fungal community of the gastrointestinal tract has recently be-
come of interest, and knowledge of its relationship with the development of obesity
is scarce. The present study aimed to evaluate the cultivable fungal fraction from the
microbiota and to analyze its relationship with obesity.
Methods: Samples were taken from 99 participants with normal weight, overweight
and obesity (n=31, 34 and 34, respectively) and were cultivated in selective medium,
and the cultivable yeasts were identified by matrix-assisted laser desorption/ioniza-
tion time-of-flight mass spectrometry. Anthropometric and biochemical measures
were also evaluated.
Results: Eutrophic, overweight and obese groups presented concentrations of 1.6,
2.16 and 2.19log10 colony-forming units g–1 yeast, respectively. Ascomycota and
Basidiomycota were the two identified phyla. At the genus level, Candida spp. showed
a relatively high prevalence, and 10 different species were detected: Candida glabrata,
Candida orthopsilosis, Candida lambica, Candida kefyr, Candida albicans, Candida
krusei, Candida valida, Candida parapsilosis, Candida utilis and Candida humilis
(with relative abundances of 71.72%, 5.05%, 21.21%, 6.06%, 29.29%, 27.27%, 8.08%,
16.16%, 1.01% and 2.02%, respectively).
Conclusions: The obese group presented a higher prevalence of Candida albicans.
Furthermore, Candida albicans, Candida kefyr and Rhodotorula mucilaginosa
showed a high positive correlation with obesity, weight gain and fat mass and showed
a negative correlation with high-density lipoprotein and lean mass, parameters re-
lated to weight loss.
KEYWORDS
anthropometry, eutrophic, fungal microorganism, intestinal mycobiota, obesity, overweight

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GARCÍA-GAMBOA et al.
functions, blood glucose regulation and energy acquisition
from food.7, 8 The diet type has an important role in the con-
figuration of the gut microbiota. For example, the Western
diet induces microbiome changes related to intestinal in-
tegrity, inflammation and the resolution of inflammation,
storage of energy, thermogenesis and homeostasis of lipids,
and the metabolism of glucose.9 In terms of quantity, bac-
teria are the most abundant microorganisms in the intesti-
nal microbiota. However, the fungal community in the gut
has begun to receive increased attention. This community
is known as the intestinal mycobiota (IMy). Although the
intestinal mycobiota represents approximately 0.03%–2.0%
of the total microorganisms in the intestinal tract, the size
of a fungal cell could be 100 times that of a bacterial cell.10
Thus, the fungal community is an important part of the in-
testinal microbiota and its functions. The presence of some
pathologies can cause intestinal disruption between micro-
organisms and the host, a condition known as intestinal dys-
biosis. Bacterial intestinal dysbiosis has been associated with
several diseases, such as diabetes,11 intestinal bowel disease12
and obesity.13,14 However, the fungal microbiota and its rela-
tionship with different diseases, such as obesity, have been
poorly explored.
Different methods can be used for the identification of
fungal microorganisms in the gut, including culture, mi-
croscopy, sequencing and matrix-assisted laser desorption/
ionization time-of-flight (MALDI-TOF) mass spectrometry.
The fungal community found in the gut belongs mainly to
three phyla: Ascomycota, Basidiomycota and Zygomycota.
To date, 278 fungal species have been reported in the gut.15
The present study aimed to evaluate the composition of the
cultivable gut mycobiota and its relationship with obesity
and several nutritional aspects.
METHODS
Subjects
In total, 99 Mexican subjects (70 female and 29 male) were
classified into three groups based on their body mass index
(BMI). Group 1 included 31 eutrophic subjects (BMI=19.5–
24.9 kg m–2), group 2 included 34 overweight subjects
(BMI= 25.0–29.9kg m–2), and group 3 included 34 obese
subjects (BMI = 30.0–39.9kgm–2). Inclusion criteria com-
prised: patients aged between 20 and 50years; an absence of
any intestinal disease, diabetes or any metabolic disease; and
not consuming antibiotics, antifungal drugs, probiotics or
prebiotics during the 3months prior to the study.
Dietetic analysis
Two quantitative questionnaires were used to determine
usual dietetic intake from participants. (1) A food frequency
questionnaire (FFQ) was used to obtain food intake infor-
mation from participants over a long period.16 In each item
of the questionnaire, participants reported the frequency
and specific amount of food intake. (2) A 24-h dietary recall
provided quantitative nutritional information about food in-
take during the last 24h.17 In the present study, nutritionists
carried out all dietetic interviews.
Anthropometric assessment
An anthropometric evaluation was performed to determine
the body composition of the participants. The equipment
was used for the anthropometric assessment comprised: a
0.5-cm wide metallic flexible measure tape graded in cen-
timeters and centimeter decimals (Cescorf), a digital scale
(model UM-040; TANITA) with a capacity of 150 kg and
grading of 100g, and an adipometer (Slim Guide) graded
in millimeters.18 Anthropometric measures included: weight
(kg); height (cm); skin folds of tricipital, bicipital, iliac crest
and subscapular (mm); and circumference of hip, waist, arm
and wrist (cm).19 All measurements were taken by an an-
thropometrist certified by the International Society for the
Advancement of Kinanthropometry (ISAK).20
Biochemical analysis
After 8 h of fasting, a sample of blood was extracted from
each participant to evaluate the metabolic status. The ele-
ments analyzed were: serum iron, ferritin, transferrin, iron
fixing capacity, total proteins, albumin, globulin, total
bilirubin, direct bilirubin, indirect bilirubin, alanine ami-
notransferase, aspartate aminotransferase, glutamic gamma
transferase, lactate dehydrogenase, cholesterol, high-density
lipoprotein, low-density lipoprotein, very low-density lipo-
protein, triglycerides, glucose, ureic nitrogen, creatinine and
uric acid.
Yeast growth by plate culture
Fecal samples (1g) from each participant were diluted with
a solution (9ml) of phosphate-buffered saline (0.85%) to ob-
tain a 1:10 dilution. One hundred microliters from each dilu-
tion was placed separately in Sabouraud Dextrose medium/
agar (Difco), potato dextrose agar/medium (Difco) and yeast
extract peptone dextrose agar (Sigma-Aldrich), and chlo-
ramphenicol was added at a concentration of 250 mg L–1.
Samples were incubated at 28 and 37°C for 48h.15 Colony-
forming units (CFUs) were quantified.
Yeast identification by MALDI-TOF
mass spectrometry
Yeast identification was carried out by mass spectrometry
utilizing the MALDI-TOF technique. Yeasts were isolated
from the feces as described above and fresh colonies were

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THE INTES TINAL MYCOBIOTA AND ITS R ELATIONSHIP W ITH OVERWEIGHT, OBESITY A ND
NUTRITIONAL ASPECTS
taken from agar plates, placed in duplicate in analysis plate
wells and allowed to dry at room temperature. The wells
were covered with 1.0 μl of matrix (saturated solution of
alpha-cyano-4-hydroxycinnamic acid [Sigma-Aldrich] in
50% acetonitrile and 2.5% trifluoroacetic acid). Readings
were obtained with a Microflex LT mass spectrometer and
Flex Control, version 3.4 (Bruker Daltonics). The spectrom-
eter was calibrated using a protein extract from Escherichia
coli (Bruker Bacterial Test Standard). Identification criteria
were: score ≥2=identification at the species level; a score
between 1.7 and 1.9=identification at the genus level; a score
<1.7=no identification.21
Statistical analysis
The mean ± SD were calculated for clinical, anthropomet-
ric, and dietetic variables. Additionally, one-way analysis
of variance (ANOVA) was used to determine whether there
were any significant differences in the means of socio-de-
mographic, anthropometric, clinic and dietetic variables,
as well as the mean count of yeast and filamentous isolated
between the groups of subjects (eutrophic, overweight and
obese). Furthermore, linear discriminant analysis (LDA) is
a method of classifying samples into categorical dependent
values (or groups, in this case, the three groups of subjects)
using linear combinations of several predictors (variables)
such that the between-group variance is maximized relative
to the within-group variance. In this study, the discrimi-
nation functions were used to analyze the yeast variations
between eutrophic, overweight and obese subjects. Both the
classification matrix and the linear discriminant functions
were calculated. In this case, the LDA was used to identify
a linear combination of the yeast populations that would
allow us to determine the groups of subjects (eutrophic,
overweight, and obese) to which each observation belongs.
The classif ication matrix shows the proportion of values cor-
rectly assigned to each season. The absolute value of each co-
efficient of the linear discriminant functions is related to the
importance of the variable to classify an observation. The
analysis was improved by adding standardized coefficients
for linear discriminant functions and the scatterplot of the
scores for these functions, providing an interpretation of the
influence of some variables to classify the observations.22,23
All statistical analyses were performed using Rstudio, ver-
sion 1.2 (https://rstud io.com/produ cts/rstudio).
Characteristics
Groups of participants
pvalue
Eutrophic
(n=31)
Overweight
(n=34)
Obese
(n=34)
n31 34 34
Gender (female/male) 20/11 25/9 26/8
Average age (years)
(mean±SD)
27.83±5.39 34.76±11.82 34.67±10.50 0.00653*
Average BMI (kgm–2) 22.32±1.75 27.15±1.20 34. 50±4.91 <2×10−16 *
Waist circumference (cm) 75.19±6.78 85.31±7.85 99.75±13.64 9.58×10−16*
Hip circumference (cm) 96.64±4.14 102.59±3.92 114.99±11.85 2.44×10−1 5*
Waist-hip ratio 0.77±0.06 0.83±0.83 0.86±0.08 0.0000227*
Fat mass (kg) 15.61±4.92 21.46±4.56 31.97±7.71 <2×10−16*
Lean mass (kg) 19.24±6.90 20.12±5.64 20.14±4.68 0.778*
Transferrin (mgdl–1) 247. 06±44.48 267.88±28.67 262.85±34.16 0.0586706
ALT/GPT (UL–1) 24.11±11.62 33.41±18.56 38.38±21.86 0.0071761*
GGT (UL–1) 11.32±6.35 26.88±18.53 25.38±15.44 4.73×10−5*
Total cholesterol (mgdl–1) 162.94±24.94 170.56±24.81 165.59±29.20 0.498
LDL (mgdl–1) 95.95±22.42 112.01±22.99 104.14±22.48 0.020*
VLDL (mgdl–1) 14.77±6.89 17.13±9.29 18.29±9.67 0.362
HDL (mgdl–1) 54.36±12.25 43.34±6.59 41.32±6.34 3.09×10−8*
Triglycerides (mgdl–1) 82.84±37.67 122.24±41.89 149.76±94.85 3.03×10− 4*
Glucose (mgdl–1) 82.19±26.87 77.03±3.01 81.00±8.27 0.387
BUN (mgdl–1) 15.0±2.90 14.83±3.28 12.82±4.25 0.017*
Ureic acid (mgdl–1) 4.98±1.0 5.84±1.06 5.76±1.54 0.017*
Values represent the mea n±SD.
Abbrevi ations: ALT/GPT, alanine t ransaminase enz yme; BMI, body mass index ; BUN, blood urea nit rogen; GGT,
gamma-glutamyl transferase enzy me; HDL, high- density lipoprotein; LDL , low-density lipoprotein; VLDL, ver y
low-density lipoprotei n.
*Values with statistically significant difference (analy sis of variance , p<0.05).
TABLE 1 Socio-demographic,
anthropometric and clinical characteristics of
the participants

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GARCÍA-GAMBOA et al.
RESULTS
The socio-demographic characteristics of the 99 partici-
pants are shown in Table 1. All groups consisted mainly of
women. The mean±SD age was 32.42±10.17years. There
were statistically significant differences in the anthropomet-
ric measurements between the three groups. Fat mass in-
creased in agreement with BMI according to the eutrophic,
overweight and obese groups (15.61±4.92, 21.46±4.56 and
31.97±7.71kg, respectively). Measurements for BMI, waist
circumference, hip circumference and waist–hip ratio grad-
ually increased from eutrophic to obese participants.
In the present study, the mean count of yeast was 1.6,
2.16 and 2.19log10CFUg–1 feces, and the mean count of fil-
amentous fungi was 1.09, 1.38 and 1.60log10CFUg–1 feces
in eutrophic, overweight and obese participants, respectively
(Table 2). From 162 fungal colonies isolated, 71 were identi-
fied and classified into 16 genera or species. In addition, 56%
of isolates were unable to be identified. It was not possible to
identify the filamentous fungi; they were only cultivated and
reported as CFUg–1 feces.
Only two fungal phyla from the cultivable mycobiota
were found; the predominant phylum was Ascomycota, of
which four different genera were observed (Candida spp.,
Geotrichum spp., Kazachstania spp. and Pichia spp.), fol-
lowed by the phylum Basidiomycota, of which two genera
were observed (Rhodotorula spp. and Cryptococcus spp.).
Only one species was observed for most of the genera. Wit hin
the genus Candida, 10 species were identified: Candida
glabrata (71.72%), Candida orthopsilosis (5.05%), Candida
lambica (21.21%), Candida kefyr (6.06%), Candida albicans
(29.29%), Candida krusei (27.27%), Candida valida (8.0 8%),
Candida parapsilosis (16.16%), Candida utilis (1.01%) and
Candida humilis (2.02%). Two different species within the
genus Geotrichum spp. were observed: Geotrichum silvicola
(16.6%) and Geotrichum candidum (7.07%) (Table 3).
The differences between the groups (eutrophic, over-
weight and obese) were further evaluated using a discrim-
inant analysis with the data set comprising the counts of 16
yesat species. Two discriminant functions were estimated
and he standardized coefficients for the linear discriminant
functions are presented on Table 4. The absolute value of
each coefficient is related to the importance of the variable
in classifying an observation to the different groups. In the
first discriminant function, C. albicans and G. silvicola had
the most significant coefficients, with values of 0.62 and
0.47, respectively. This function discriminated well between
groups, and higher values of this function were associated
with the obese group. Candida krusei also had one of the
most significant coefficients of −0.47, and this variable was
associated with overweight. Figure 1 shows the scatterplot
for the scores of the two linear discriminant functions. A
pattern existed for the data in the three different groups, w ith
overlapping zones, meaning that the discriminant functions
can discriminate between groups. Observations of the obese
group had higher scores for the first discriminant function,
followed by cases in the overweight group, and then the eu-
trophic group had lower scores (with an overlap for the last
two groups).
The phyla Ascomycota and Basidiomycota were found
in all three populations. For Ascomycota, C. albicans,
C. lambica and G. silvicola were predominant in the obese
groups compared to the eutrophic and overweight groups.
Candida kefyr, C. valida, G. candidum and P. kluyveri were
found only in obese subjects. Candida glabrata was pre-
dominant in eutrophic subjects compared to obese and
overweight subjects. We also found the presence of C. ortho-
psilosis, C. utilis and Kazachstania exigua only in eutrophic
subjects. For Basidiomycota, Rhodotorula mucilaginosa was
predominant in obese subjects compared to eutrophic and
overweight subjects. Meanwhile, Cryptococcus liquefaciens
was found only in the eutrophic group (Table 3).
Table 5 shows the dietary information collected using two
dietetic questionnaires; these questionnaires encompassed
the information for a long (6 months) and a short period
(24h). The results showed a difference in the intake for some
dietetic groups; for example, total kilocalories (kcal), carbo-
hydrate kcal, protein kcal, complex carbohydrates, meat kcal,
and vegetable kcal. These nutrients increased from eutro-
phic to the obese group. There was an increase in the intake
of lipids, dairy products and simple carbohydrates in obese
people; however, no significant difference was observed.
Biochemical parameters were significantly different be-
tween the groups (Table 1). Alanine transaminase and gam-
ma-glutamyl transferase enzymes, low-density lipoprotein,
triglycerides, and ureic acid (38.38 ± 21.86, 25.38 ± 15.44,
104.14±22.48, 149.76±94.85 and 5.76±1.54, respectively)
were the parameters with the highest concentrations in the
obese group compared to the eutrophic group, showing a
significant difference (p<0.05). Other parameters, such as
transferrin, aspartate transaminase and lactate dehydroge-
nase enzymes, were increased in subjects with overweight
and obesity. However, these parameters did not reach a sta-
tistically significant difference. High-density lipoprotein
and blood urea nitrogen showed a high presence in the eu-
trophic group (54.36± 12.25 and 15.0±2.90, respectively)
Eutrophic (n=31) Overweight (n=34) Obese (n=34) pvalue Groups
Tot al f ungi 1.88±0.7 2.18±0.6 2.89±0.729 0.01 a,b,b
Yea s t 1.6±0.7 2.16±0.6 2.19±0.74 0.01 a,b,b
Filamentous 1.09±0.1 1.38±0.5 1.60±0.11 0.2 a,a,a
Values represent the mea n ±SD. Log10 of CFUg–1 fecal sample were c alculated to nor malize and present the d ata.
The same lowercase le tters represent homogeneous groups (a,b).
TABLE 2 Yeast and filamentous counts
isolated from subjects

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THE INTES TINAL MYCOBIOTA AND ITS R ELATIONSHIP W ITH OVERWEIGHT, OBESITY A ND
NUTRITIONAL ASPECTS
and these parameters were diminished in the overweight
and obese groups (p<0.05).
Figures 2, 3 and 4 show the correlation among anthropo-
metric, biochemical and dietetic parameters with cultivable
mycobiota from eutrophic, overweight and obese subjects.
Anthropometric data showed a high positive and negative
correlation with the yeasts; weight, BMI, waist circumfer-
ence, hip circumference and waist–hip ratio fat mass showed
a high positive correlation with Basidiomycota, Rhodotorula
spp., C. albicans, C. lambica, C. kefyr and C. valida (p<0.05).
Alanine transaminase, aspartate transaminase, low-density
lipoprotein and very low-density lipoprotein showed a high
positive correlation with G. candidum (p < 0.05). High-
density lipoprotein showed a high positive correlation with
Kazachstania spp. and K. exigua (p<0.05). Lipid consump-
tion showed a high positive correlation with Kazachstania
spp., K. exigua, G. silvicola and C. parapsilosis. Kcal showed
a positive correlation with G. candidum, and simple carbo-
hydrate consumption showed a positive correlation with
C. lambica (p <0.05).
DISCUSSION
Most of the fungal species inhabiting the gastrointestinal
tract are commensals and opportunistic pathogens that
could turn into potential threats depending on strain vir-
ulence traits and the status of the host immune system.24
From this perspective, to discover a pathogenic infection, it
is crucial to define exactly which species are normally pre-
sent in each body niche. In the present study, obese subjects
showed a higher yeast CFU compared to subjects with nor-
mal weight. This is consistent with another study25 in which
subjects with obesity displayed a higher presence of yeasts
compared to eutrophic and overweight subjects. Another
study demonstrated that the increase in the total yeast count
in feces was negatively correlated with the expression of the
PPA Rα gene, for which activation is involved in the decrease
in fat accumulation.26 Therefore, it is possible to hypothesize
that the high presence of yeasts and PPARα gene expression
is related to weight and body fat control. The results of the
present study show that cultivable IMy belonged to the phyla
Ascomycota and Basidiomycota, and Ascomycota presented
a higher prevalence in the three groups. This is in agree-
ment with other studies that have reported Ascomycota and
Fungi
Eutrophic
(n=31)
Overweight
(n=34)
Obese
(n=34) Tot a l
Relative
abu ndan ce (%)
Candida glabrata 21 11 19 71 51.52
Candida orthopsilosis 5 0 0 5 5.05
Candida lambica 0 8 13 21 21.21
Candida kefyr 0 0 6 6 6.06
Candida albicans 3 3 23 29 29.29
Candida krusei 11 16 027 27.27
Candida valida 0 0 8 8 8.08
Candida parapsilosis 8 8 0 16 16.16
Candida utilis 1 0 0 1 1.01
Candida humilis 0 2 0 2 2.02
Rhodotorula mucilaginosa 4 2 14 20 20.20
Cryptococcus liquefaciens 2 0 0 2 2.02
Geotrichum silvicola 3 1 12 16 16.16
Geotrichum candidum 0 0 7 7 7.07
Kazachstania exigua 7 0 0 7 7. 0 7
Pichia kluyveri 0 0 1 1 1.01
TABLE 3 Quantification of yeast species
isolated from fecal samples of participants
TABLE 4 Comparison of linear discriminant analysis between
groups (eutrophic, overweight and obese)
Yea s ts LD1 LD2
Candida glabrata 0.10 0.20
Candida orthopsilosis −0.27 0.65
Candida lambica 0.33 −0.48
Candida kefyr 0.18 0.10
Candida albicans 0.63 0.22
Candida krusei −0.48 −0.45
Candida valida 0.18 −0.02
Candida parapsilosis −0.26 −0.04
Candida utilis −0.16 0.26
Candida humilis −0.01 −0.04
Rhodotorula mucilaginosa 0.25 0.46
Cryptococcus liquefaciens −0.06 0.25
Geotrichum silvicola 0.21 − 0.01
Geotrichum candidum 0.48 0.13
Kazachstania exigua −0.35 0.65
Pichia kluyveri 0.09 −0.08
Proport ion of trace: LD1 0.76, LD2 0.23.
Abbrevi ations: LD, linea r discriminant.

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GARCÍA-GAMBOA et al.
Basidiomycota as the two dominant phyla in the human
IMy.27–30 Other studies showed a high proportion of fungal
species belonging to Ascomycota compared to those be-
longing to Basidiomycota.10,25,31 Candida spp. was the most
prevalent genus in the present study. Hoffmann et al.32 in-
vestigated the relationship between diet and fungal commu-
nity in the gut and found that Candida spp. was positively
correlated with carbohydrate consumption, mainly simple
sugars, similar to the present study. However, this correla-
tion was not statistically significant. Furthermore, we found
that obese and overweight subjects consumed more carbo-
hydrates than eutrophic subjects. Borgo et al.33 reported
that the presence of Candida spp. could be related to the
breakdown of starch in carbohydrate foods, leading to the
release of simple sugars in the gut that are in turn fermented
by bacteria (e.g., Prevotella and Ruminococcus). Regarding
bacterial gut microbiota, much evidence shows the role of
two specific bacterial phyla in obese subjects (low abun-
dance of Bacteroidetes and high abundance of Firmicutes).1
The Firmicutes–Bacteroidetes ratio can be modified under
controlled weight and diet, especially with a high lipid con-
tent.34 Related to this, Hoffmann et al.32 and Mukherjee
et al.35 reported a negative correlation between Candida spp.
and Bacteroidetes. This negative correlation is related to an
FIGUR E 1 Linear discriminant (LD) analysis yeast cultivable community from feces of eutrophic, overweight and obese subjects.
Characteristics
Groups of participants
pvalue
Eutrophic
(n=31)
Overweight
(n=34) Obese (n=34)
Kilocalories (kcal) 1612.42±293.08 1636.38±302.15 1858.42±302.52 0.0017 *
Carbohydrates (kcal) 787.1±205.56 800.71±211.50 951.53±197.46 0.0020*
Protein (kcal) 343.68±86.40 337.76±93.38 407.06±111.06 0.0073*
Lipid (kcal) 481.65±200.06 497.91±159.90 499.62±140.82 0.8943
Simple carbohydrate (kcal) 38.71±28.86 71.76±51.73 73.53±49.70 0.1506
Complex carbohydrate (kcal) 748.39±201.89 728.94±180.58 886.18±186 0. 0016*
Dairy products (kcal) 63.29±40.75 51.88±31.76 50.82±30.41 0.6764
Meat (kcal) 173.42±82.56 179.53±77.10 218.24±68.16 0.0373*
Veg etab le ( kcal) 106.97±39.04 106.35±49.98 137.41±45.66 0.0075*
Values represent the mea n ±SD.
*Values with statistically significant difference (analy sis of variance , p<0.05).
TABLE 5 Dietetic characteristics
obtained from the participants

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7
THE INTES TINAL MYCOBIOTA AND ITS R ELATIONSHIP W ITH OVERWEIGHT, OBESITY A ND
NUTRITIONAL ASPECTS
increase in Candida spp. and a decrease in Bacteroidetes,
showing a Bacteroidetes profile similar to that in obesity.
The results of the present study showed a high presence
of Candida in overweight and obese subjects compared to
eutrophic subjects. Thus, it is possible that overweight and
obese groups will show a high amount of Firmicutes and a
low presence of Bacteroidetes. To confirm this, it will be nec-
essary to study gut bacteria in future assays.
Candida albicans is considered part of the indigenous
intestinal microbiota, and it has also been recognized that
its colonization is widely related to the aggravation of in-
flammation. Several studies have reported that subjects
with obesity showed different degrees of inflammation.36 A
high presence of C. albicans in feces from obese subjects was
present compared to eutrophic and overweight subjects. Ng
et al.37 reported that the main carbon source of C. albicans is
glucose; therefore, it is possible that the presence of C. albi-
cans is a result of the high sugar intake of obese participants.
Furthermore, C. albicans showed a highly positive correla-
tion with obesity parameters and metabolic diseases (weight,
BMI, hip and waist circumference, waist–hip ratio and fat
mass) and showed a negative correlation with high-density
lipoprotein and lean mass, comprising parameters related to
good health.38 Additionally, in the LDA, C. albicans showed
the most significant coefficient.
Candida glabrata was present in all groups; thus, it is
possible that this yeast does not belong to a specific group.
The relative abundance of this yeast was higher than that
of other species. Interestingly, eutrophic subjects showed a
higher prevalence of C. glabrata. However, most of the pre-
vious studies showed that this microorganism is associated
with infections39 and hospitalized patients.40,41 Further
studies are necessary to understand the role of C. glabrata in
Mexican IMy or metabolism.
Rhodotorula mucilaginosa was abundantly present in
the obese group. In the gut microbiota, this microorgan-
ism is considered as an opportunistic pathogen.42 It is
widespread in the environment, and its transmission to
humans occurs mainly through air and food. However,
its growth in the gastrointestinal tract is suppressed be-
cause the growth conditions in the body are not optimal.
Rhodotorula mucilaginosa shows a massive presence in the
gastrointestinal tract when there is a disruption in the in-
testinal microbiota or dysbiosis.43 One particular reason
for this disruption is treatment with azoles, which creates
an advantage for these azole-resistant fungi. Hospitalized
patients treated with azoles (fluconazole and posacon-
azole) presented a high presence of R. mucilaginosa
compared to those treated with amphotericin B and flu-
cytosine, which are the most active antifungal agents.44,45
FIGUR E 2 Correlation among cultivable
gut mycobiota of eutrophic, overweight and
obese groups with anthropometric variables.
Letters in parenthesis (P, C, O, F, G and S)
indicate phylum, class, order, family, genus
and species, respectively.

8
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GARCÍA-GAMBOA et al.
It is important to note that high amounts of R. mucilag-
inosa are not alarming. By contrast, when this microor-
ganism colonizes the human gastrointestinal tract, it may
produce some nutrients, such as proteins, lipids, folate and
carotenoids.43 Rhodotorula yeasts are capable of metabo-
lizing short-chain fatty acids such as acetic, propionic and
FIGUR E 3 Correlation among cultivable
gut mycobiota of eutrophic, overweight and
obese groups with biochemical variables.
Letters in parenthesis (P, C, O, F, G and S)
indicate phylum, class, order, family, genus
and species, respectively.
FIGUR E 4 Correlation among cultivable
gut mycobiota of eutrophic, overweight, and
obese groups with dietetic variables. Letters
in parenthesis (P, C, O, F, G and S) indicate
phylum, class, order, family, genus and
species, respectively.

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9
THE INTES TINAL MYCOBIOTA AND ITS R ELATIONSHIP W ITH OVERWEIGHT, OBESITY A ND
NUTRITIONAL ASPECTS
butyric acids. Therefore, it is feasible that a massive pres-
ence of Rhodotorula spp. in the gut could lead to a shortage
of these bacterial products. Rhodotorula spp. are capable
of producing lipids by utilizing glucose,46 acetic acid and
propionic acid. Additionally, this genus may produce satu-
rated and unsaturated fatty acids.47 With this information,
it is possible to hypothesize that the higher presence of
R. mucilaginosa in the obese group plays an important role
in obesity development and its positive correlation with fat
mass. Some of the fatty acids produced by Rhodotorula spp.
are palmitic and stearic acids (16:0 and 18:0, respectively);
Aro et al.48 reported that these fatty acids are important
in the decrement of high-density lipoprotein. In the pres-
ent study, the obesity group presented the lowest amount
of HDL. Additionally, adipocyte tissue utilizes palmitic
and stearic acid for lipogenesis.49 Moreover, a study re-
ported the cholesterolemic effect of palmitic acid,50 and
the presence of this molecule was high in obese subjects.
It is unclear how the IMy affects host lipid metabolism, as
has been reported with intestinal bacteria.51 Furthermore,
Rodríguez et al.10 related Rhodotorula to metabolic abnor-
malities associated with lipid alteration. Thus, it is pos-
sible to hypothesize that IMy, especially Rhodotorula, is
capable of modulating fatty acid production, energy and
the nutritional status of the host, contributing to the de-
velopment of obesity. This could be related to the results
in the present study because Rhodotorula spp. showed a
positive correlation with weight, BMI, hip and fat mass.
Therefore, with these results and the role of Rhodotorula
spp. in lipid metabolism, it is feasible to expect that this
yeast is related to obesity.
Candida kefyr was identified only in obese subjects and
showed a positive correlation with BMI and tricipital, sub-
scapular, suprail iac subcutaneous fat. Furthermore, carbohy-
drate consumption, mainly simple sugars, showed a positive
correlation with C. kefyr, although it did not reach statistical
significance. This is similar to another study that reported
the presence of C. kefyr in oral microbiota only in obese
subjects compared to normal-weight subjects.52 However,
other studies related the presence of C. kefyr to a decrease in
obesity or weight loss.26 Candida kefyr is another yeast that
is capable of modifying the Bacteroides–Firmicutes ratio,32
and this modification can change the development of obe-
sity.53 Thus, it is possible that C. kefyr may affect the control
weight.
Candida lambica was present only in the overweight
and obesity groups. Furthermore, LDA showed a high
coefficient in these subjects. Candida lambica showed a
high positive correlation with variables related to weight
gain in subjects, including weight, BMI, subcutaneous fat
(tricipital, bicipital and suprailiac) and fat mass accumu-
lation. In addition, C. lambica presented a high positive
correlation with the waist–hip ratio, which is a parameter
related to android central obesity (defined as central or
abdominal obesity with excess distribution of body fat in
the abdomen). This type of obesity is characterized by sev-
eral health risks, such as hypertension, cardiometabolic
diseases and insulin resistance.54 Fraberger et al.55 re-
ported that C. lambica is capable of metabolizing simple
sugars, such as glucose, fructose and sucrose. According
to the dietetic results, overweight and obese participants
consumed high amounts of carbohydrates. It has been re-
ported that a percentage of carbohydrates escape digestion
in the small intestine and reach the colon, and these car-
bohydrates could be consumed by this microorganism.56
Scott et al.57 reported that 40g of carbohydratesreach the
gut microbiota every day, and this value is dependent on
the carbohydrate intake in the diet. Therefore, in high car-
bohydrate diets, such as those consumed by overweight
and obese groups, more carbohydrates will be in the colon,
and, with this, the growth of microorganisms, such as
C. lambica, may occur. It is important to emphasize that,
in the present study, K. exigua and C. orthopsilosis were
only present in eutrophic subjects, whereas C. valida and
G. candidum were only present in obese subjects. No re-
ports have clearly indicated the role of these two yeasts in
the gastrointestinal tract; thus, it is important to investi-
gate the functions of these microorganisms in IMy in more
detail.
In conclusion, the results of the present study demon-
strate the differences between cultivable IMy in eutrophic,
overweight and obese Mexican subjects. Obese subjects
showed higher CFU values, whereas the eutrophic group
showed lower CFU values. Ascomycota and Basidiomycota
were the two phyla found from the identified fungal mi-
croorganisms, and Ascomycota was the most prevalent.
Candida spp. was the genus that showed the highest num-
ber of species. C. albicans was the most prevalent yeast
present in the obese subjects. Candida albicans, C. kefyr
and R. mucilaginosa were yeasts that showed a high pos-
itive correlation with obesity, weight gain and fat mass,
and also showed a negative correlation with high-density
lipoprotein and lean mass, comprising parameters related
to weight loss. On the other hand, K. exigua and C. ortho-
psilosis were found only in eutrophic subjects. These re-
sults were obtained from culture-dependent techniques.
Therefore, we propose studying the gut mycobiota of these
populations with molecular techniques aining to investi-
gate the noncultivable fungal community and its relation-
ship with obesity.
TRANSPARENCY DECLARATION
The lead author affirms that this manuscript is an honest,
accurate and transparent account of the study being re-
ported. The lead author affirms that no important aspects of
the study have been omitted and that any discrepancies from
the study as planned have been explained.
ACKNOWLEDGMENTS
We thank the financial support granted by CONACYT
(CVU number 662891)
CONFLICT OF INTERESTS
The authors have no conflicts of interest.

10
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GARCÍA-GAMBOA et al.
AUTHOR CONTRIBUTIONS
Ricardo García-Gamboa: Study and design, development
& methodology, collection of data, data analysis/interpre-
tation, written all section of the manuscript. Manuel R.
Kirchmayr: Development & methodology and manuscript
revision. Misael Sebastián Gradilla-Hernández: manu-
script revision. Vicente Pérez-Brocal: Development &
methodology and manuscript revision. Andrés Moya: Study
and design, manuscript revision. Marisela González-Avila:
Study and design, development & methodology, collection of
data, data analysis/interpretation.
PEER REVIEW
The peer review history for this article is available at https://
publo ns.com/publo n/10.1111/jhn.12864.
ORCID
Ricardo García-Gamboa https://orcid.
org/0000-0002-7226-3315
Misael S. Gradilla-Hernández https://orcid.
org/0000-0002-8236-4400
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How to cite this article: García-Gamboa R,
Kirchmayr MR, Gradilla-Hernández MS, Pérez-
Brocal V, Moya A, González-Avila M. The intestinal
mycobiota and its relationship with overweight,
obesity and nutritional aspects. J Hum Nutr Diet.
2021;00:1–11. https://doi.org/10.1111/jhn.12864