microorganisms

Article
Maternal Diet May Modulate Breast Milk Microbiota—A Case
Study in a Group of Colombian Women
Diana C. Londoño-Sierra 1 , Victoria Mesa 1,2 , Nathalia Correa Guzmán 1 , Laura Bolívar Parra 3 ,
Olga I. Montoya-Campuzano 3 and Sandra L. Restrepo-Mesa 1, *

1 Food and Human Nutrition Research Group, School of Nutrition and Dietetics, Antioquia University,
Medellín 050010, Colombia; [email protected] (D.C.L.-S.);
[email protected] (V.M.); [email protected] (N.C.G.)
2 Physiopathologie et Pharmacotoxicologie Placentaire Humaine Microbiote Pré & Postnatal (3PHM), INSERM,
UMR-S 1139, Université Paris Cité, 75006 Paris, France
3 Probiotics and Bioprospecting Research Group, Faculty of Sciences, National University of Colombia,
Medellín 050034, Colombia; [email protected] (L.B.P.); [email protected] (O.I.M.-C.)
* Correspondence: [email protected]; Tel.: +57-3006085609

Abstract: There is increasing evidence that the diet and nutritional status of women during pregnancy
and lactation can modulate the microbiota of their milk and, therefore, the microbiota of the infant. An
observational, descriptive, and cross-sectional study was carried out in a group of lactating women.
Dietary intake during gestation and the first trimester of lactation was evaluated, and the microbiota
was analyzed by 16S ribosomal RNA (rRNA) sequencing using the Illumina platform. Globally,
Streptococcus spp. (32%), Staphylococcus spp. (17.3%), Corynebacterium spp. (5.1%) and Veillonella spp.
(3.1%) were the predominant bacterial genera. The consumption of simple carbohydrates in gestation
(rho = 0.55, p ≤ 0.01) and lactation (rho = 0.50, p ≤ 0.01) were positively correlated with Enterobacter
spp. In lactation, a negative correlation was observed between the intake of simple carbohydrates
and the genus Bifidobacterium spp. (rho = −0.51 p ≤ 0.01); furthermore, a positive correlation was
Citation: Londoño-Sierra, D.C.; identified between the intake of folic acid and Akkermansia spp. (rho = 0.47, p ≤ 0.01). Amplicon
Mesa, V.; Guzmán, N.C.;
sequence variants (ASVs) associated with the delivery mode, employment relationship, the baby’s
Bolívar Parra, L.;
gender, birth weight, the Body Mass Index (BMI) of the breastfeeding woman, and gestational weight
Montoya-Campuzano, O.I.;
gain were recovered as covariates in a linear mixed model. The results of this research showed that
Restrepo-Mesa, S.L. Maternal Diet
May Modulate Breast Milk
the maternal nutritional status and diet of women during gestation and lactation could modulate the
Microbiota—A Case Study in a microbiota of breast milk.
Group of Colombian Women.
Microorganisms 2023, 11, 1812. Keywords: breast milk microbiota; maternal nutrition; nutritional status
https://doi.org/10.3390/
microorganisms11071812

Academic Editor: Min Yue

1. Introduction
Received: 15 May 2023 The role of the human microbiota in healthy growth and development during the first
Revised: 9 June 2023 years of life has been related to its beneficial metabolic and structural functions, the regula-
Accepted: 16 June 2023
tion of immunity and systemic inflammation, as well as its influence on the somatotrophic
Published: 14 July 2023
axis, which has regulated the production of growth factors such as the insulin-like growth
factor 1 (IGF1) and growth hormone, as well as energy and nutritional metabolism [1,2].
Moreover, the first interaction of microorganisms with humans is fundamental for immuno-
Copyright: © 2023 by the authors.
logical, metabolic, and systemic adaptation; the first 1000 days of life are considered a
Licensee MDPI, Basel, Switzerland.
decisive immunological window for health in the later stages of life [3].
This article is an open access article Due to its enormous metabolic capacity, the microbiota is considered essential for life,
distributed under the terms and with great influence on health and disease. The population composition of the microbiota
conditions of the Creative Commons is particular and exhibits its own characteristics in each individual; therefore, it varies
Attribution (CC BY) license (https:// according to genetics, the mode of birth, the type of feeding in the early years, habitual diet,
creativecommons.org/licenses/by/ the use of probiotics, exposure to antibiotics, interactions with the environment, among
4.0/). other factors [4]. It is estimated that about 25–30% of the infant microbiota has its origin in

Microorganisms 2023, 11, 1812. https://doi.org/10.3390/microorganisms11071812 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 1812 2 of 19

breast milk [2], in which a central bacteriome composed of nine genera, Staphylococcus spp.,
Streptococcus spp., Serratia spp., Pseudomonas spp., Corynebacterium spp., Ralstonia spp.,
Propionibacterium spp., Sphingomonas spp., and Bradyrhizobium spp. has been identified [5]. A
recent study conducted in Colombia on breast milk samples from women donors identified
that the milk microbiota contained commensal microorganisms, including, among them,
lactic acid bacteria with probiotic potential using culture methods [6]; however, no studies
have been developed in a national context to identify how a woman’s diet modulates the
microbiota of her milk.
It has been documented that breast milk microbiota is modulated by several factors
such as diet and weight [5]. Regarding maternal feeding, the consumption of carbohydrates,
fiber and vegetable proteins could influence the abundance of Staphylococcus spp., Bifidobac-
terium spp., and Lactobacillus spp. [7], and associations have also been found between the
intake of polyunsaturated fatty acids and the genus Bifidobacterium spp. [8]. Micronutri-
ents such as calcium and vitamin B2 have been positively associated with Veillonella spp.
abundance [9] and vitamin C intake with a higher abundance of Staphylococcus spp. [8].
In relation to the Body Mass Index (BMI) and gestational weight gain, changes in breast
milk microbiota have also been identified. The BMI of lactating women has been negatively
related to the genus Bacteroides spp. [9] and gestational weight gain has shown a positive
relationship with alpha diversity in breast milk microbiota [10]. It has been described
that excess maternal weight could generate changes in the milk’s metabolome and in the
microbiota: mechanisms that could be involved in the risk of infant obesity [11–13].
The connection between nutritional status, a woman’s diet during gestation and
lactation, and the microbiota of breast milk highlights the need to go deeper into this
subject in order to identify aspects that, from a dietary and nutritional point of view, could
be modified in favor of the microbiota, which can contribute to favoring the health of
the mother-child binomial in the short, medium and long term. The complexity of breast
milk and the factors that contribute to its composition are essential aspects that must
be taken into account in order to expand our knowledge and design strategies targeting
breast milk microbiota to influence maternal and infant health. The objective of this study
was to analyze the effects of food and nutritional status during gestation and the first
trimester of lactation on the microbiota of breast milk in a group of healthy lactating
women in Colombia.

2. Materials and Methods

An observational, descriptive, and cross-sectional study was carried out on a group of
breastfeeding women who had prenatal care in two health institutions in eastern Antio-
quia, Colombia.

2.1. Subjects
The sample consisted of 30 women in their first trimester of lactation who were selected at
convenience and who had a prenatal medical history in the referenced institutions (Figure 1).
The criteria for the selection of mothers were the following: breastfeeding women
between 18 and 39 years of age who had a singleton pregnancy, without diseases or
complications during gestation and postpartum (anemia, diabetes, hypertensive disorders,
among others), with adequate BMI or overweight, at least three prenatal controls, with
food security at home according to the Latin American and Caribbean Food Security
Scale (ELCSA) [14], who had a full-term newborn, and who exclusively breastfed their
child. Women with obesity (BMI > 30 kg/m2 ) or thinness (<18.5 kg/m2 ) and women
who, during gestation, lactation, and up to 30 days prior to data collection, chronically
consumed medications or other substances such as antibiotics, antidepressants, laxatives,
corticosteroids, cigarettes, alcohol, proton pump inhibitors, and probiotics were excluded.
Microorganisms 2023, 11, x FOR PEER REVIEW 3 of 20
Microorganisms 2023, 11, 1812 3 of 19

Figure 1. Flow diagram summarizing the identification and selection of participants and the method-
Figure 1. Flow diagram summarizing the identification and selection of participants and the meth-
ology process.
odology process.
The participants were identified at the health institution; then, their medical history
The criteriaand
was reviewed, for the selection
if they met the of mothers
inclusionwere the following:
criteria, they were breastfeeding
contacted to sign women the
between 18 and 39 years of age who had a singleton
informed consent form and to schedule two visits for data collection.pregnancy, without diseases or com-
plications during gestation and postpartum (anemia, diabetes, hypertensive disorders,
among others), with
2.1.1. Collection adequate BMI Data
of Anthropometric or overweight, at least three prenatal controls, with
food security
The women’s weight and height Latin
at home according to the were American
measuredand usingCaribbean Food and
digital scales Security Scale
a portable
(ELCSA)
measuring [14], who
rod, andhad a full-term
with these data newborn,
the BMIand (BMI who exclusively
= kg/m 2 ) was breastfed
calculated, their child.
which ac-
Women
cordingwithto the obesity
World(BMI
Health> 30Organization
kg/m2) or thinness
(WHO), (<18.5
waskg/m
2) and women who, during
classified as adequate (≥18.5 to
gestation,
<25 kg/mlactation, and up to
2 ) or overweight (≥3025days
kg/m prior
2 toto data
<30 collection,
kg/m chronically
2 ). To collect consumed med-
anthropometric infor-
ications
mation in the gestational stage, the participants’ clinical history was askedcorticosteroids,
or other substances such as antibiotics, antidepressants, laxatives, for in terms of
cigarettes, alcohol, proton
their pregestational pump
maternal weightinhibitors,
and theand probiotics
weight reported were excluded.
in the prenatal controls. Total
Thegain
weight participants were identified
during gestation at the health
was determined by theinstitution;
differencethen, theirthe
between medical history
pregestational
was reviewed,
weight and theand lastifweight
they met the inclusion
reported criteria,history.
in their clinical they were contacted
Adequate to sign the
gestational in-
weight
formed
gain was consent form and
considered to schedule
based two visits
on the adjusted for data
allowed collection.
ranges for the Pregestational Body
Mass Index (PBMI) according to the references of the Institute of Medicine of the United
2.1.1.
StatesCollection of Anthropometric
(IOM): underweight 12.5 and Data
18 kg; normal weight 11.5–16 kg; overweight 7 and
11.5 The
kg [15];
women’sweight gain was
weight and classified
height were as excessive
measured whenusing itdigital
exceeded
scalesthese
and ranges and
a portable
insufficientrod,
measuring whenanditwith
did not
these reach
datathetheminimum
BMI (BMI expected.
= kg/m2) was Newborn weight
calculated, whichandaccord-
length
datatowere
ing taken; Health
the World birth weight was classified
Organization (WHO), as was
insufficient at 2500–2999
classified as adequate g and adequate
(≥18.5 to <25
between
2 3000 and 3999 g [16].
kg/m ) or overweight (≥25 kg/m to <30 kg/m ). To collect anthropometric information in
2 2

the gestational stage, the participants’ clinical history was asked for in terms of their
2.1.2. Evaluation of Food Consumption
pregestational maternal weight and the weight reported in the prenatal controls. Total
weight Two
gain24during
h recalls (R24h) were
gestation applied to each
was determined participant,
by the differenceusing the adjusted
between multi-step
the pregestational
technique on non-consecutive days, and were distributed during the week:
weight and the last weight reported in their clinical history. Adequate gestational weight a procedure
necessary
gain to adjust forbased
was considered intra-on
and inter-individual
the adjusted allowedvariability
ranges[17]. To determine
for the the amount
Pregestational Body
of food intake, a set of models, geometric figures, and an album of photographs
Mass Index (PBMI) according to the references of the Institute of Medicine of the United with
life-size utensils were used, which are validated for Colombia [18].
States (IOM): underweight 12.5 and 18 kg; normal weight 11.5–16 kg; overweight 7 and
11.5 kgTo [15];
identify the aspects
weight gain wasrelating to nutrition
classified duringwhen
as excessive pregnancy, a quantitative
it exceeded frequency
these ranges and
of consumption was applied, which was composed of 81 foods selected
insufficient when it did not reach the minimum expected. Newborn weight and length from the consump-
tion reported in pregnant women in the Food and Nutrition Profile of the Department
data were taken; birth weight was classified as insufficient at 2500–2999 g and adequate
of Antioquia 2019 [19]. This included foods such as legumes, fruits, and vegetables for
between 3000 and 3999 g [16].
Microorganisms 2023, 11, 1812 4 of 19

their contribution of soluble and insoluble fiber [20]; sources of saturated, unsaturated, and
polyunsaturated fatty acids [21] such as lard, vegetable margarine, butter, industrialized
sauces, vegetable oil, olive, canola, nuts, and seeds; fermented foods such as yogurt [22];
animal proteins such as beef, pork, chicken, fish, and eggs; industrialized meats; and foods
high in simple and ultra-processed carbohydrates [23].
To test the estimation of nutrients from the quantitative frequency of foods, a con-
cordance test was performed between the designed frequency and the R24h in a group
of seven pregnant women belonging to the prenatal control program of one of the ref-
erence institutions to whom both instruments were applied for subsequent analysis. To
estimate these differences, the Wilcoxon signed-rank test, the Hodges-Lehmann test and
95% confidence intervals (95% CI) were applied; to evaluate this correlation, the Biserial
Rank Correlation Coefficient (95% CI) was applied and to evaluate the concordance, the
Concordance Correlation Coefficient was also used (95% CI) (Table S1). The data collected
for this test were not included in the study.

2.1.3. Food Consumption Analysis

For nutrient analysis, the R24h was processed in the Dietary Intake Evaluation software
(EVINDI v5) of the School of Nutrition and Dietetics of the University of Antioquia [24],
and the database obtained was migrated into the Personal Computer Software for Intake
Distribution Estimation (PC-SIDE v1.0) [25], which is available at the Department of
Statistics at Iowa State University, Ames IA (United States). For each nutrient, the Estimated
Average Requirement (EAR) established in the Recommendations for Energy and Nutrient
Intake for the Colombian population was used as a cut-off point. For macronutrients,
the Acceptable Macronutrient Distribution Range (%AMDR) was recommended in the
national guidelines [26].
The prevalence of the risk of deficiency was accompanied by summary measures
such as the minimum, maximum, percentiles, mean, and standard deviation, which were
adjusted in PC-SIDE v1.0 [25]. The contribution of nutrients of interest was obtained:
calories, proteins, total fat, saturated, monounsaturated, polyunsaturated, cholesterol, total
and simple carbohydrates, dietary fiber, zinc, calcium, iron, magnesium, vitamins: B1, B2,
B3, B5, B6, B9, B12, A, and C. Data processing and analysis were performed in SPSS v25,
EVINDI v5.0 and PC-SIDE v1.0 software.

2.2. Collection of Breast Milk Samples

The samples were collected between 8 and 10 a.m., manually, and from the breast
opposite to the last suckling of the newborn or from the breast opposite to the one from
which the baby was suckling. The nipple and the surrounding area were cleaned with sterile
gauze and 0.5% chlorhexidine. Between 15 and 20 mL of milk were collected, discarding
the first drops, and the milk was deposited in sterile tubes free of RNAses and DNAses
(Corning Incorporated, Corning, NY, USA). After the collection process, the milk samples
were transported in a cooler with dry ice to the Food and Human Nutrition Research
Laboratory, University of Antioquia, Colombia (transport time less than 1 h), where they
were stored at −80 ◦ C for subsequent DNA extraction.

2.3. Extraction, Quantification and Sequencing of Barcoded Amplicons on the Illumina

MiSeq Platform
The total genomic DNA extraction was performed from 6 to 10 mL of breast milk using
the GeneJET Genomic DNA Purification kit (Thermo Scientific) at the molecular biology
laboratory of the Faculty of Agricultural Sciences, University of Antioquia, Colombia. The
extracted DNA was quantified using the 260/280 optical density ratio by UV absorbance
methods (NanoDrop, Thermo Scientific, Wilmington, NC, USA). Hypervariable regions
V3-V4 of the 16S ribosomal ribonucleic acid (rRNA) gene were amplified using 1 µL of
DNA (25 ng on average). A Polymerase Chain Reaction (PCR)was performed in 27 cycles
within the following reaction conditions: 95 ◦ C for 20 s, 55 ◦ C for 20 s, and 72 ◦ C for 20 s.
Microorganisms 2023, 11, 1812 5 of 19

The primers used were Bakt_341F: CCTACGGGGGNGGCWGCAG and Bakt_805R: GAC-

TACHVGGGGGTATCTAATCC, and each sample was assigned a unique 6-base pair (bp)
barcode. Barcoded PCR products were purified from triplicate reactions with an agarose
gel band purification kit (Illustra GFX PCR dna and gel Band Purification Kit, GE Health-
care, UK). Equimolar concentrations of PCR amplicons were quantified by fluorometric
methods (Qubit 3.0—Thermo Fisher Scientific, Waltham, MA, USA). Purified amplicons
were pooled in equimolar amounts (~50 ng per sample) for library preparation. Sequencing
was performed using the Illumina MiSeq paired-end platform (2 × 300 base pairs) with
100,000 reads for each library (Macrogen, Korea).

2.4. Analysis of Microbiota Data

These sequences were demultiplexed, thus removing the primer sequences and associ-
ated barcodes. The bioinformatics analysis of sequences was performed in QIIME2 (Quan-
titative Insights into Microbial Ecology) v2019.7 software [27]. The DADA2 method [28]
was used to detect and correct sequencing noise, remove chimeric sequences, and cluster
sequences into amplicon sequencing variants (ASVs). To classify these sequences according
to their taxonomic information, the qiime feature-classifier plugin was employed using
the Vsearch alignment method [29] with the SILVA v138 database [30] at a 99% sequence
identity. Subsequently, the BIOM (Biological Observation Matrix Data) table (frequency
table of each ASV with its taxonomic assignment), the phylogenetic tree, and the metadata
of samples with the information of variables under study were imported for analysis in the
RStudio v1.1.453 software [31].

2.5. Statistical Analysis

For the descriptive analysis of sociodemographic, gestational, anthropometric, and
food consumption characteristics, absolute and relative distributions and summary indi-
cators such as the arithmetic mean and standard deviation were used. To compare the
diversity and richness of the bacterial community, alpha diversity was analyzed using four
indices: the Chao1 index, which estimates the richness of taxa in a community [32], the
Shannon index, which allows an evaluation of the heterogeneity of a community based
on the number of species present and their relative abundance [33]; Simpson’s inverse
index, also known as the dominance index, which allows measurements of the richness
of organisms [34] and the PD index, which describes diversity based on phylogenetic
distances [35]. Comparisons between these groups were performed using non-parametric
tests: the Wilcoxon rank test or Kruskal–Wallis. For beta diversity, principal coordinate
analysis (PCoA) was performed to identify a clustering pattern of microbial compositions
as a function of the variables of interest using permutation-based methods (PERMANOVA,
permuted multivariate analysis of variance, using the Adonis2 library) for weighted and
unweighted UniFrac distances [36]. Diversity analyses were performed using the Phy-
loseq [37] and Microbiome [38] packages of the Rstudio v4.1.2 software [31].
Correlations between the most abundant and literature-reported bacterial genera and
nutrient intake values were explored using Spearman’s correlation coefficient, which was
visualized by a heatmap in the RStudio program using the Corrplot package [39]. Through
a mixed linear model (LMM), the association of the most abundant ASVs transformed
logarithmically with the individual variables registered in the metadata and were evaluated
using the RStudio’s lme4 and nlme packages [40,41]. Later, with the ASVs that showed
a significant association (p < 0.05), the model was adjusted to control the effects of other
variables so that the resulting variation explained was independent of other variables and
not subject to confusion by the correlated variables; each variable was the fixed factor and
the others entered as covariates.
The study followed the ethical considerations established in the Declaration of Helsinki
and was validated by the Bioethics Committee of the Faculty of Dentistry of the University
of Antioquia: concept No. 66-2020, Act No. 10 of 2020. Informed consent was obtained
from all participants.
Microorganisms 2023, 11, 1812 6 of 19

2.6. Data Availability Statement

Sequence reads were deposited in the European Nucleotide Archive (ENA) via the
project number PRJEB59523.

3. Results
3.1. Sociodemographic, Gestational and Anthropometric Characteristics
The average age of the lactating women was 25 ± 6 years; 73% had the presence of a
partner, 37% had finished higher education, 57% lived in a rural area, and 53% belonged
to the subsidized health regime. Of the total number of participating women, 60% did
not plan their pregnancy, 53% had between 1 and 2 children, 80% attended more than six
prenatal check-ups, and 43% were first-time mothers.
In relation to the anthropometric characteristics, the average weight of the lactating
women was 60.8 ± 7.9 kg, 60% presented a normal BMI %, and the majority presented a
height ≥ 1.55 m (63%). In relation to gestation, most of the women started with a BMI in
adequacy (70%), and at the end, the average weight gained was 12.2 ± 3.6 kg; 43% presented
inadequate weight gain due to deficiencies, and 13% presented with an inadequate gain
due to excess. More than half of them had a vaginal delivery (77%); the newborns presented
an average of 3299 ± 275 g and 49.8 ± 1.7 cm at birth, were breastfed in the first hour of life
87%, and were male 63% (Table 1).

Table 1. Sociodemographic, gestational, and anthropometric characteristics of the breastfeeding women.

Variable n %
Sociodemographics
Marital Status
With partner 22 73.3
Without partner 8 26.7
Level of schooling *
Elementary school 1 3.3
Secondary School 7 23.3
High school 11 36.7
Higher education 11 36.7
Health Regime *
Contributory 14 46.7
Subsidized 16 53.3
None 10 33.3
Zone of residence
Urban 13 43.3
Rural 17 56.7
Gestational
Pregnancy planning
Yes 12 40.0
No 18 60.0
Previous pregnancies
No pregnancies 13 43.3
1–2 16 53.3
≥3 1 3.3
No. of prenatal checkups
≤5 6 20.0
6–8 17 56.7
≥9 7 23.3
Type of delivery
Vaginal 23 76.7
Cesarean section 7 23.3
Anthropometric
Pregestational BMI (Kg/m2 )
Adequate 21 70.0
Thin 1 3.3
Overweight 8 26.7
Breastfeeding BMI (Kg/m2 )
Adequate 18 60.0
Overweight 12 40.0
Gestational weight gain
Adequate 13 43.3
Deficit 13 43.3
Excess 4 13.3
Data presented as n (%). * Educational level and health status were classified according to national guidelines.
Microorganisms 2023, 11, 1812 7 of 19

3.2. Nutrient Intake

The mean adjusted energy intake was 2185 calories (Standard Deviation (SD) = 399),
the prevalence of risk of deficiency in usual energy intake was 43% (SD = 0.11), and the
risk of excess was 16% (SD = 0.12). Regarding the consumption of macronutrients, the
prevalence of the risk of deficiency in the usual intake of proteins was 99% (SD = 0.03), the
consumption above the reference value of total fat (>35% AMDR) was 3% (SD = 0.12) and
of total carbohydrates (>65% AMDR) was 1% (SD = 0.06). Regarding the consumption of
nutrients of interest, the mean adjusted intake of cholesterol was 493 mg (SD = 145), the
consumption above the reference value (>10% ADMR) of saturated fat was 86% (SD = 0.18),
and for simple carbohydrates was 72% (SD = 0.10); 97% of the women did not reach the
recommended fiber intake (Table 2).

Table 2. Distribution and adequacy of energy intake, the percentage of individuals with intakes
above and below the Acceptable Macronutrient Distribution Range (% AMDR) and prevalence of
risk of deficiency for the usual intake of protein, vitamins and minerals.

<% >%
Prevalence of Adjusted Adjusted Adjusted Percentiles Adjusted
Reference Reference
Nutrients Deficiency Minimum Maximum Mean
Value * Value *
% (SD) (SD) ***
% (SD) % (SD) 5 25 50 75 90

Calories (Kcal) 42.8 (0.11) 16.0 (0.12) 890 3419 1528 1917 2186 2455 2695 2185 (399)
Protein (g) 50.3 (0.14) 0.2 (0.01) 98.5 (0.03) 33.3 131.3 50.0 65.0 75.0 85.0 94.0 74.8 (15)
Total fat (g) 0.2 (0.01) 3.2 (0.12) 29.4 120.3 49.0 60.1 68.7 77.9 86.8 69.4 (13.2)
Saturated fat (g) 14.02(0.184) 86.0 (0.18) 9.28 50.30 18.80 24.40 28.40 32.70 36.60 28.60 (6.12)
Monounsaturated
10.20 40.40 16.00 20.00 23.40 27.10 30.40 23.65 (4.97)
fat (g)
Polyunsaturated
5.12 28.65 9.20 11.00 12.40 14.00 15.70 12.60 (2.32)
fat (g)
Cholesterol (mg) 70 1299 277 389 480 583 686 493 (145)
Total
2.3 (0.10) 1.0 (0.06) 98.3 568.9 207.0 267.0 311.0 359.0 403.0 314.1 (68.0)
carbohydrates (g)
Simple
28.0 (0.102) 72.0 (0.10) 1.9 243.2 18.0 48.0 77.0 109.0 144.0 82.8 (48.2)
carbohydrates (g)
Dietary Fiber (g) 3.1 (0.05) ** 48.2 49.8 5.8 9.2 12.6 17.1 22.4 13.9 (6.6)
Vitamin A (ER) 49.8 (0.12) 172 13,063 371 599 903 1489 2638 1396 (1803)
Vitamin C (mg) 64.9 (0.10) 14 381 25 49 77 122 185 98 (75)
Folates (ugEFD) 87.3 (0.10) 49 929 130 202 273 368 478 299 (136)
Zinc (mg) 52.3 (0.09) 4.22 24.63 5.90 8.00 9.80 11.90 14.10 10.16 (3.00)
Calcium (mg) 30.2 (0.15) 239 2892 548 761 940 1147 1360 971 (293)
Iron (mg) 9.1 (0.10) 4.5 61.7 8.9 12.3 16.1 22.0 30.1 18.6 (9.5)
Thiamine (mg) 57.0 (0.17) 0.22 5.26 0.77 0.97 1.15 1.36 1.59 1.19 (0.30)
Riboflavin (mg) 0.6 (0.04) 0.91 7.65 1.53 1.87 2.18 2.59 3.05 2.29 (0.59)
Niacin (mg) 51.6 (0.10) 3.7 24.2 7.6 10.3 12.8 15.8 18.5 13.2 (3.9)
Panthotenic acid (mg) 18.8 (0.10) ** 2.19 11.97 3.40 4.50 5.50 6.60 7.80 5.67 (1.63)
Vitamin B6 (mg) 59.8 (0.11) 0.74 3.91 1.03 1.31 1.58 1.93 2.34 1.68 (0.51)
Vitamin B12 (ug) 7.3 (0.10) 0.78 93.56 2.20 3.90 6.90 14.30 32.30 16.18 (37.20)
Magnesium (mg) 53.3 (0.10) 83 492 163 217 260 305 349 263 (65)
Data are presented as percentages (%), standard deviation (SD), minimum and maximum values, adjusted
percentiles and adjusted mean. * Reference values for calories 90–110%; protein 14–20%AMDR; total fat
20–35%AMDR; saturated fat 10%AMDR; total carbohydrates 50–65%AMDR; simple carbohydrates 10%AMDR.
** Low risk of deficiency for dietary fiber and pantothenic acid. *** SD: standard deviation.

In relation to micronutrient intake, the highest prevalence in the risk of deficiency of

usual intake was presented for folic acid at 87% (SD = 0.10), vitamin C at 65% (SD = 0.10),
vitamin B6 at 60% (SD = 0.11), thiamine at 57% (SD = 0.17), magnesium at 53% (SD = 0.10),
zinc at 52% (SD = 0.09), niacin at 52% (SD = 0.10) and vitamin A at 50% (SD = 0.12). By
contrast, the lowest prevalence in the risk of deficiency of usual intake was presented in
iron intake 9.1% (SD = 0.10), vitamin B12 7.3% (SD = 0.10), riboflavin 0.6% (SD = 0.04) and
the prevalence of a low risk of deficiency in pantothenic acid intake was 18.8% (SD = 0.10)
as presented in Table 2. Regarding the consumption of supplements during gestation, all
participants consumed iron and folic acid supplements 97%, which was not the case with
calcium supplementation, where only 37% reported daily consumption; however, it should
be noted that due to the consumption of dairy products, the risk of calcium deficiency
was low.
Microorganisms 2023, 11, 1812 8 of 19

In the survey of the mother’s food intake during gestation, it was found that the
median energy intake was 2.553 calories (median absolute deviation, MAD = 597.5), protein
94 g (MAD = 24.5), total fat 71 g (MAD = 17.1), carbohydrates 379 g (MAD = 97), simple
carbohydrates 76 g (MAD = 36.1) and dietary fiber 24 g (MAD = 8.1). Regarding the intake
of nutrients of interest, the median intake of saturated fat was 28 g (MAD = 6.95), and
simple carbohydrates was 76 g (MAD = 36.1); for micronutrient intake, the highest median
intakes during gestation were for vitamin A intake 1394.5 ER (MAD = 443.5) and folic acid
1461.5 mg (MAD = 257) Table 3.

Table 3. Distribution of energy and nutrient intake during pregnancy.

Percentiles
Nutrients Minimum Maximum 5 25 50 75 90 Median (MAD) *
Kcal (Kcal) 1495 5895 1582.1 2036.25 2553 3163.75 4408.6 2553 (597.5)
Protein (g) 56.2 171.5 59.715 80.825 93.8 131.425 160.2 93.8 (24.5)
Total Fat (g) 37.9 135.5 45.94 58.45 71.05 93.4 113.95 71.1 (17.1)
Saturated fat (g) 15.6 56.36 17.909 22.165 28.055 36.52 47.35 28.1 (6.95)
Monounsaturated fat (g) 13.28 57.97 17.508 22.105 28.965 40.542 46.027 28.9 (8.16)
Polyunsaturated fat (g) 4.91 23.34 6.369 9.185 11.115 15.063 20.469 11.1 (2.59)
Cholesterol (mg) 108 1698 189 432.25 544.5 886.25 1174.6 544.5 (229)
Total carbohydrates (g) 145.1 1024.8 200.025 279.975 378.45 451.975 678.6 378.5 (97)
Simple carbohydrates (g) 2.6 323.9 8.905 42.55 76.2 112.85 135.34 76.2 (36.1)
Dietary Fiber (g) 11.2 63.9 12.8 20.075 23.8 37.6 42.46 23.8 (8.1)
Vitamin A (ER) 602 3761 668.7 1023.25 1394.5 1927.75 2252.8 1394.5 (443.5)
Vitamin C (mg) 109 840 124.45 222.25 254.5 437 658.1 254.5 (70)
Folates (ugEFD) 1043 2541 1130.05 1243.75 1461.5 1903.25 2114.9 1461.5 (257)
Zn (mg) 6.9 34.8 7.445 9.2 12.4 18.025 21.12 12.4 (4.4)
Fe (mg) 34.3 146.2 41.965 76.675 80.35 88.3 99.42 80.4 (5.15)
Thiamine (mg) 0.99 4.48 1.038 1.373 1.995 3.07 3.741 1.9 (0.67)
Riboflavin (mg) 1.43 6.3 1.484 2.053 2.855 4.442 5.476 2.9 (0.99)
Niacin (mg) 10.4 45.7 12.615 16.25 20.35 27.725 33.44 20.4 (5.75)
B6 (mg) 1.4 7.5 1.445 1.825 2.6 3.2 4.31 2.6 (0.65)
B12 (ug) 1.96 20.37 2.839 4.765 6.58 10.387 14.238 6.6 (2.33)
Mg (mg) 181 820 192.6 263.75 346 511 619.7 346 (97.5)
Data are presented as minimum, maximum, percentiles and the absolute deviation from the median. * MAD:
median absolute deviation.

3.3. Bioinformatic Analysis of the Milk Microbiota

After filtering, merging, and checking for chimera sequences of the 16S RNA gene,
in the V3-V4 region, from the 30 samples collected, a total of 2,940,975 sequences were
obtained. The sequencing depth of the data processed by the DADA2 method [28] ranged
from 56,744 to 126,815 sequences per sample (Table S2). Rarefaction curves showed the
number of taxa (richness or alpha diversity) as a function of the sample size or the number
of reads. Most samples reached the plateau effect, indicating that the diversity of all
sequences obtained was sampled (Figure S2).

3.4. Characterization of the Milk Microbiota

In the characterization of the microbiota of breast milk samples from lactating women
in Colombia, 25 bacterial phyla were found, the most abundant being: Firmicutes (69.5%),
Actinobacteria (10%), Proteobacteria (9.6%), and Bacteroidetes (7.6%). The bacterial phyla
identified are listed in Figure S1.
Regarding the bacterial genera, 644 were detected, with the most abundant being
Streptococcus spp. (32%), Staphylococcus spp. (17.3%), Corynebacterium spp. (5.1%) and
Veillonella spp. (3.1%). Ten genera were identified with a relative abundance between 1.2%
and 2.6%, Bacteroides spp. (2.6%), Lactobacillus spp. (2.4%), Bacillus spp. (1.9%), Rothia spp.
(1.8%), Methylobacterium spp. (1.6%), Clostridum sensu stricto 1 spp. (1.5%), Pseudomonas spp.
(1.4%), Fusobacterium spp. (1.4%), Gemella spp. (1.3%) and Prevotella spp. (1.3%) (Figure 2).
 in the V3-V4 region, from the 30 samples collected, a total of 2940,975 sequences were
obtained. The sequencing depth of the data processed by the DADA2 method [28] ranged
from 56,744 to 126,815 sequences per sample (Table S2). Rarefaction curves showed the
number of taxa (richness or alpha diversity) as a function of the sample size or the number
of reads. Most samples reached the plateau effect, indicating that the diversity of all se-
Microorganisms 2023, 11, 1812 9 of 19
quences obtained was sampled (Figure S2).

3.4. Characterization of the Milk Microbiota

Differences in Alpha
In and Beta Diversity in
the characterization of Relation to the Variables
the microbiota of breast under Study from lactating
milk samples
women in Colombia, 25 bacterial phyla were found, the most abundant being: Firmicutes
A trend toward higher bacterial richness and diversity was observed in the milk
(69.5%), Actinobacteria (10%), Proteobacteria (9.6%), and Bacteroidetes (7.6%). The bacte-
samples from women with a C-section delivery (Chao 1 p = 0.022; PD p = 0.03) (Figure 3A)
rial phyla identified are listed in Figure S1.
and those who hadRegarding
a direct employment relationship or through a family member (Chao1
the bacterial genera, 644 were detected, with the most abundant being
≤ 0.01) (Figure
p = 0.029; PD pStreptococcus 3B). No differences
spp. (32%), Staphylococcus spp. were found
(17.3%), according to
Corynebacterium the(5.1%)
spp. area and
of Veil-
residence, maternal age, level of schooling, the number of previous pregnancies, or the
lonella spp. (3.1%). Ten genera were identified with a relative abundance between 1.2% sex
of the infant; however,
and 2.6%,itBacteroides
was identified that breast
spp. (2.6%), milk microbiota
Lactobacillus spp. (2.4%),from women
Bacillus with male
spp. (1.9%), Rothia spp.
infants showed a tendency to have a greater richness and diversity (Chao1
(1.8%), Methylobacterium spp. (1.6%), Clostridum sensu stricto 1 spp. (1.5%),p = 0.74; PD
Pseudomonas
p = 0.06) (Figure 3C).
spp. In newborns
(1.4%), withspp.
Fusobacterium insufficient birth weight,
(1.4%), Gemella there
spp. (1.3%) andwas also a spp.
Prevotella tendency
(1.3%) (Fig-
ure 2). and diversity in the microbiota compared to those with an adequate
for a lower richness
birth weight (Chao1 p = 0.99; PD p = 0.84) (Figure 3D).
100
R e la tive a bu n da n ce (% )

75

50

25

0
S1
S2
S3

S1
S1
S1
S1
S1
S1
S1
S1
S1
S1

S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
S3
S4
S5
S6
S7
S8
S9

0
1
2
3
4
5
6
7
8
9

0
1
2
3
4
5
6
7
8
9
0
Streptococcus Rothia g__un_f__Muribaculaceae Parabacteroides Plesiomonas
Staphylococcus Methylobacterium Bradyrhizobium Blautia Actinomyces
Corynebacterium 1 Clostridium sensu stricto 1 Terrisporobacter Escherichia Shigella Others
Veillonella Pseudomonas g__un_f__Lachnospiraceae Acinetobacter
Bacteroides Fusobacterium Prevotella 7 Pantoea
Lactobacillus Gemella Ruminococcaceae UCG 014 Haemophilus
Bacillus Prevotella Trueperella Bifidobacterium

Figure 2. Bacterial genera

Figure identified
2. Bacterial in breast
genera milk
identified in microbiota.
breast milk microbiota.

Differences in Alpha and Beta Diversity in Relation to the Variables under Study
 0.029; PD p ≤ 0.01) (Figure 3B). No differences were found according to the area of resi
dence, maternal age, level of schooling, the number of previous pregnancies, or the sex o
the infant; however, it was identified that breast milk microbiota from women with male
infants showed a tendency to have a greater richness and diversity (Chao1 p = 0.74; PD p
= 0.06) (Figure 3C). In newborns with insufficient birth weight, there was also a tendency
Microorganisms 2023, 11, 1812 10 of 19
for a lower richness and diversity in the microbiota compared to those with an adequate
birth weight (Chao1 p = 0.99; PD p = 0.84) (Figure 3D).

Figure 3. Alpha diversity indices of breast milk microbiota. (A) Type of delivery; (B) Employment
relationship; (C) Baby’s
Figuregender; (D)
3. Alpha Birth weight
diversity indices where G1:
of breast birth
milk weight of(A)
microbiota. 2700 g and
Type <3000 g;
of delivery; (B)G2:
Employmen
3000 g and 3500 g; G3: >3500 g.(C) Baby’s gender; (D) Birth weight where G1: birth weight of 2700 g and <3000 g; G2
relationship;
3000 g and 3500 g; G3: >3500 g.
According to the nutritional status by anthropometric indicators, a tendency to have a
higher alpha diversityAccording to the
through the nutritional
Shannon indexstatus
andby anthropometric
InvSimpson indicators,
index a tendency
was observed in to have
women with an adequate BMI without statistical differences (Shannon p = 0.22; InvSimpson observed
a higher alpha diversity through the Shannon index and InvSimpson index was
p = 0.18) (Figure in women
Microorganisms 2023, 11, x FOR PEER REVIEW
4A); a lowerwith an adequate
richness BMI without
and diversity statistical
was also differences
observed in those(Shannon
women p11=of0.22
20
InvSimpson p = 0.18) (Figure 4A); a lower richness and diversity was also observed in those
who had excessive gestational weight gain (Chao1 p = 0.51; PD p = 0.96) (Figure 4B).
women who had excessive gestational weight gain (Chao1 p = 0.51; PD p = 0.96) (Figure
4B).

Figure 4.indices
Figure 4. Alpha diversity Alpha diversity
of breast indices of breast milk
milk microbiota microbiota
in relation in relation to anthropometric
to anthropometric indicators ofindicators
of nutritional status. (A) BMI of breastfeeding woman; (B) Gestational weight gain.
nutritional status. (A) BMI of breastfeeding woman; (B) Gestational weight gain.
The principal coordinate analysis (PCoA) was performed to identify a clustering pat-
tern of microbial composition based on the weighted UniFrac distances, where the dis-
tance represented the difference between microbial communities, taking into account phy-
logenetic distances and unweighted UniFrac where they are not taken into account. Based
on the weighted UniFrac distances, the beta diversity of breast milk microbiota presented
Microorganisms 2023, 11, 1812 11 of 19

Figure 4. Alpha diversity indices of breast milk microbiota in relation to anthropometric indicators
of nutritional status. (A) BMI of breastfeeding woman; (B) Gestational weight gain.
The principal coordinate analysis (PCoA) was performed to identify a clustering pat-
The principal coordinate analysis (PCoA) was performed to identify a clustering pat-
tern of microbial composition based on the weighted UniFrac distances, where the distance
tern of microbial composition based on the weighted UniFrac distances, where the dis-
represented the difference between microbial communities, taking into account phyloge-
tance represented the difference between microbial communities, taking into account phy-
neticlogenetic
distances and unweighted
distances UniFrac
and unweighted where
UniFrac they
where areare
they not taken
not takeninto
intoaccount. Based on
account. Based
the weighted UniFrac distances, the beta diversity of breast milk microbiota
on the weighted UniFrac distances, the beta diversity of breast milk microbiota presented presented
differences according
differences to to
according gestational
gestationalweight
weight gain (PERMANOVA
gain (PERMANOVA p =p 0.033).
= 0.033).TheThe pairwise
pairwise
beta-diversity comparisons
beta-diversity comparisons between
betweengroups showedsignificant
groups showed significant overall
overall differences
differences across
across
the groups, with
the groups, higher
with distance
higher distancedissimilarities amonggroups
dissimilarities among groups belonging
belonging to inadequacies
to inadequacies
by deficiencies
by deficiencies andand excess,
excess, whichwas
which was observed
observed when
whencompared
compared to the reference
to the adequate.
reference adequate.
The variables
The variables under
under analysisexplained
analysis explained the
the variability
variability ofofthe microbiota
the microbiota in 63.5% for the
in 63.5% for the
weighted
weighted UniFrac
UniFrac distances
distances and11.8%
and 11.8%for
for the
the unweighted
unweighted UniFrac
UniFrac distances
distances (Figure 5). 5).
(Figure

Figure 5. Principal coordinate analysis (PCoA) to identify a clustering pattern of microbial composi-
Figure 5. Principal coordinate analysis (PCoA) to identify a clustering pattern of microbial compo-
tion based on weighted
sition based and unweighted
on weighted UniFrac
and unweighted distances
UniFrac and
distances according
and accordingtotogestational
gestationalweight
weight gain.
gain.beta
Pairwise Pairwise beta diversity
diversity dissimilarities
dissimilarities assessed
assessed by Unifrac
by Unifrac distancesand
distances andrepresented
represented by
byaabox-
boxplot
(* p <plot (* ****
0.05, p < 0.05, **** p < 0.0001,
p < 0.0001, Reference
Reference groupgroup = Adequate).
= Adequate).

3.5. Relationship of Nutrient Intake during Lactation with the Microbiota of Breast Milk
The intake of macro and micronutrients during lactation showed positive correlations
with the microbiota, which meant that the higher the intake of a nutrient, the higher the
abundance of a bacterial genus. In relation to macronutrient intake, a positive correlation
was identified between the consumption of simple carbohydrates and Enterobacter spp.
(rho = 0.50, p ≤ 0.01); on the other hand, the intake of total fat (rho = 0.39, p = 0.03), saturated
fat (rho = 0.38, p = 0.03), and monounsaturated fat (rho = 0.42, p = 0.02) showed a positive
correlation with the genus Eubacterium spp. (Figure 6).
 tion was identified between the consumption of simple carbohydrates and Enterobacter
spp. (rho = 0.50, p ≤ 0.01); on the other hand, the intake of total fat (rho = 0.39, p = 0.03),
saturated fat (rho = 0.38, p = 0.03), and monounsaturated fat (rho = 0.42, p = 0.02) showed
a positive correlation with the genus Eubacterium spp. (Figure 6).
Microorganisms 2023, 11, 1812 12 of 19

Correlations between
Figure6.6.Correlations
Figure between breast milk
milk microbial
microbialgenera
generaand
andnutrient
nutrientintake during
intake duringlactation and
lactation
and gestation. Heatmaps of Spearman rank correlations. Significant correlations (* p <
gestation. Heatmaps of Spearman rank correlations. Significant correlations (* p < 0.05, ** p ≤ 0.01, 0.05, ** p ≤
0.01, *** p ≤ 0.001). Blue circles represent positive correlations, whereas red circles show
*** p ≤ 0.001). Blue circles represent positive correlations, whereas red circles show negative correla- negative
correlations.
tions. Note: Note: the Fletter
the letter and RF on
andthe
R left
on the
sideleft sideheat
of the of the
mapheat map
refers to refers to Food Consumption
Food Consumption Frequency
Frequency and 24 h recall, respectively.
and 24 h recall, respectively.

Regarding
Regardingmicronutrient
micronutrientintake,
intake,aapositive
positivecorrelation
correlationwaswasidentified
identifiedbetween
betweenfolic folic
acid
acid intake and Akkermansia spp. (rho = 0.47, p ≤ 0.01); between B complex vitaminssuch
intake and Akkermansia spp. (rho = 0.47, p ≤ 0.01); between B complex vitamins such
asasB1
B1(rho
(rho==0.45,
0.45,pp==0.01),
0.01),B2B2(rho
(rho==0.51,
0.51,pp≤≤0.01),
0.01),B3
B3(rho
(rho==0.46,
0.46,pp≤≤0.01)
0.01)and
andthe
thegenus
genus
Gemella
Gemellaspp.;
spp.;asaswell
wellasasvitamin
vitaminAA intake
intakeandandthethe
genera
generaBifidobacterium
Bifidobacteriumspp. (rho
spp. = 0.36,
(rho p
= 0.36,
=p0.047), Corynebacterium
= 0.047), Corynebacterium spp.spp.
(rho (rho
= 0.43,
= p0.43,
= 0.01)
p =and RuminococcusUCG.009
0.01) and Ruminococcus UCG.009 spp. (rhospp. =
0.49,
(rhop=≤0.49,
0.01)p(Figure
≤ 0.01)6).
(Figure 6).
There
There were negativeororinverse
were negative inversecorrelations
correlationswhere
wherethethehigher
higherthetheconsumption
consumptionofofaa
nutrient,
nutrient,the
thelower
lowerthetheabundance
abundanceofofaabacterial
bacterialgenus
genusororininthe
theopposite
oppositedirection.
direction.Among
Among
these,
these,several
severalmacro
macroandandmicronutrients
micronutrientspresented
presentednegative
negativecorrelations
correlationswithwiththe
thegenus
genus
Aerococcus
Aerococcusspp.;
spp.;among
amongthis thiswas
wasthe
theconsumption
consumptionof ofthe
thetotal
totalprotein
protein (rho −0.45,p p= =0.01),
(rho ==−0.45, 0.01),
total carbohydrates (rho = −0.47, p ≤ 0.01), cholesterol (rho = −0.49, p ≤ 0.01), dietary fiber
(rho = −0.61, p ≤ 0. 01), vitamin A (rho = −0.46, p = 0.01), vitamin C (rho = −0.58, p ≤ 0.01),
folic acid (rho = −0.51, p ≤ 0.01), pantothenic acid (rho = −0.49, p ≤ 0.01), magnesium
(rho = −0.58, p ≤ 0.01) (Figure 6).
Saturated fat intake showed a negative correlation with several bacterial genera:
Corynebacterium 1 spp. (rho = −0.49, p ≤ 0.01), Cutibacterium spp. (rho = −0.36, p = 0.047),
Escherichia-Shigella spp. (rho = −0.51, p ≤ 0.01) and Lachnospiraceae NK4A136
(rho = −0.36, p = 0.049); a negative correlation was also observed between the intake
of simple carbohydrates and the genus Bifidobacterium spp. (rho = −0.51 p ≤ 0.01), as well
as dietary fiber and Enterobacter spp. (rho = −0.36, p = 0.047) (Figure 6).
Microorganisms 2023, 11, 1812 13 of 19

3.6. Relationship of Nutrient Intake during Gestation with Breast Milk Microbiota
Positive correlations were identified between the intake of macronutrients such as
simple carbohydrates (rho = 0.55, p ≤ 0.01), total carbohydrates (rho = 0.39, p = 0.02),
saturated fat (rho = 0.39, p = 0.03) and the total protein (rho = 0.3, p = 0.04) with the
genus Enterobacter spp. Saturated fat intake was also positively correlated with the genus
Halomonas spp. (rho = 0.41, p = 0.02). Regarding micronutrient intake, positive correlations
were observed between zinc intake and Pseudomonas spp. (rho = 0.38, p = 0.03), vitamin C
and Rothia spp. (rho = 0.44, p = 0.01) (Figure 6).
Protein intake (rho = −0.46, p = 0.01) and saturated fat (rho = −0.52, p ≤ 0.01) were
negatively correlated with Bifidobacterium spp.; on the other hand, dietary fiber intake was
negatively correlated with Ruminiclostridium 9 spp. (rho = −0.40, p = 0.02), Ruminococ-
caceae UCG.005 (rho = −0.39, p = 0.03), Ruminococcaceae UCG.014 (rho = −0.39, p = 0.02)
and Ruminococcus 1 spp. (rho = −0.47, p ≤ 0.01). Other correlations were identified and are
shown in Figure 6.

3.7. Association of Breast Milk Microbiota with Variables

Through the mixed linear model, the demographic, biochemical, and clinical variables
of the study were integrated with the most abundant ASVs. High specificity was identified,
indicating that ASVs could be strictly associated with each variable. The employment
relationship, birth weight, and gestational weight gain showed the highest number of
associated ASVs. After adjusting the model (with the ASVs that showed a significant
association (p < 0.05), the model was fitted to control the effects of other variables), a
significant decrease in the Peptococcus spp. was found in vaginal delivery compared to
C-section delivery; a significant increase in Eubacterium spp. was found in the overweight
category during lactation compared to adequate BMI; Aquabacterium spp., Acinetobacter,
Lawsonella spp., and Chryseobacterium spp. were found to be in a higher proportion in
samples from women with inadequate gestational weight by deficiencies compared to
adequacies (Table 4).

Table 4. Interaction between the most frequent ASVs (p < 0.05) and analyzed variables.

Variable Groups 1 ASVS Associated Top ASVs Associated p-Value Adjusted p-Value
0.8707
Bacillus 0.0036
(−0.21)
Delivery mode C-Section/Vaginal 2
6 × 10−4
Peptococcus 0.0041
(−1.09 for Vaginal)
0.0190
Actinobacillus 0.026612
(−2.03)
0.0086
Mogibacterium 0.030953
(−1.18)
Employment 0.0136
relationship Contributive/Subsidized 10 Prevotellaceae NK3B31 group 0.033343
(−2.00)
0.1172
Corynebacterium 0.038361
(−1.20)
0.3832
Gemella 0.039113
(−0.93)
Microorganisms 2023, 11, 1812 14 of 19

Table 4. Cont.

Variable Groups 1 ASVS Associated Top ASVs Associated p-Value Adjusted p-Value
0.0125
Prevotella2 0.040020
(−2.19)
0.1062
Micrococcus 0.040481
(−1.57)
0.007
Kocuria 0.046419
(−2.08)
0.0495
Phascolarctobacterium 0.048834
(−1.56)
0.036
Ruminococcus1 0.050555
(−1.69)
0.014
(−1.83)
Acinetobacter 0.003059
0.0962
Baby’s gender Female/Male 3 Staphylococcus 0.028668
(−0.95)
Alloprevotella 0.047407
0.0882
(1.37)
<0.001
Brachybacterium 0.000319
(−0.98)
Birth weight G1/G2,G3 47
<0.005
Clostridium sensu stricto 5 0.001973
(−3.14)
0.0254
Eubacterium 0.027936 (0.70 for Overweight)
BMI of the Aquabacterium 0.040497 0.0697
Adequate/Overweight 3
breastfeeding woman Acinetobacter 0.049149 (−1.63)
0.0813
(−1.31)
0.008, 0.7101
(2.99 for Inadequate
by deficit, 0.43 for
Inadequate by excess)
Aquabacterium 0.00180
Adequate/Inadequate 0.0090, 0.3281
Acinetobacter 0.01088
Gestational weight by deficiencies, (2.03, 1.04)
5 Bradyrhizobium 0.03515
gain Inadequate by 0.0965, 0.0633
Lawsonella 0.03831
excess (0.93, 1.53)
Chryseobacterium 0.04149
0.0164, 0.2976
(2.34, −1.41)
0.0267, 0.8725
(1.86, 0.18)
1 Linear mixed model and adjustment by interactions. The reference group for the comparison is highlighted
in bold. The most representative ASVs associated with each variable included in the model are shown; after
adjusting for all factors, the last column shows the p-value and in parentheses the sense of the interaction.

4. Discussion
The results of this research show that maternal nutritional status and the diet of women
during gestation and lactation could modulate the microbiota of breast milk. Excessive
gestational weight gain, low micronutrient intake, and a high intake of simple sugars and
saturated fat could impact the content of bacterial genera that is of interest for infant health,
while the consumption of micronutrients of interest, such as folates, could contribute to the
presence of bacteria with probiotic potential.
Four dominant phyla were found in this sample in the order: Firmicutes, Actinobacte-
ria, Proteobacteria, and Bacteroidetes. Investigations such as that of Urbaniak et al. [42]
in breast milk from 39 Canadian Caucasian women and Togo et al. [43], in a systematic
review, included a total of 15,849 samples from 38 countries and reported Proteobacteria
and Firmicutes as dominant phyla in breast milk, while Actinobacteria and Bacteroidetes
occurred in lower relative abundances.
A total of 644 bacterial genera were identified, which is higher than the genera reported
by Zimmermann et al. [44] in a systematic review that included 44 studies and 2655 women
Microorganisms 2023, 11, 1812 15 of 19

from 20 countries, in which 590 genera were identified. The results at the genus level were
consistent with those found by Padilhaet al. [8] in the milk samples from Brazilian women,
who reported Streptococcus, Staphylococcus, and Corynebacterium as dominant genera. This
was also reported by Kim SY et al. [45] in Korean women; specifically, Staphylococcus
spp. and Streptoccocus spp. were reported as the dominant genera [46], which could
suggest that regardless of the geographical location of the lactating woman, both genera
are represented in this fluid and their colonization could be linked to the retrograde flow
from the oral cavity of the infant [47]. Some Streptococcus spp. and Staphylococcus spp.
species have been associated with infant health by preventing the colonization of pathogens
such as Staphylococcus aureus, a risk factor for sepsis in newborns, through mechanisms
including the release of peptides with antimicrobial properties and hydrogen peroxide
production [48,49], which is of interest to the promotion of breastfeeding in all areas,
including the clinical setting.
The genera Lactobacillus spp. and Bifidobacterium spp. are important for infant health.
In this study, Lactobacillus spp. presented a relative abundance of 2.4%, which is higher
than that reported in breast milk samples from Brazilian women (0.06%) [8] and lower
than that reported in European (3.2%) [7] and Canadian women (3%) [42]. The presence
of Lactobacillus spp. in breast milk is important for its probiotic potential in relation to
health from the first years of life [50]. Breastfed infants, unlike those who receive infant
milk formula, present a microbiota richer in Lactobacillus spp. and bifidobacterial; however,
in this study, the abundance of bifidobacteria was low in agreement with that documented
in other works (1.4%) [7].
Several factors are involved in the modulation of the breast milk microbiota. The
relationship between microbiota and type of delivery is controversial since some studies
have not reported significant differences [10,42,51] while other reports have; one example
is Khodayar et al. [52], who identified higher abundances of total bacteria in the colostrum
and transitional milk of women who had a cesarean delivery, and Cortés et al. [7] who
determined a higher microbial richness in the milk of women who had undergone a
cesarean delivery, producing results consistent with those found in the present study.
Some hypotheses about the origin of the microbiota of breast milk have been doc-
umented: the retrograde translocation of bacteria from the oral cavity of the infant, the
mother’s skin, the use of breast pumps, the oro-mammary route, and the entero-mammary
route, the latter explaining how some bacteria present in the maternal gut and how their
metabolites could reach the mammary gland during late pregnancy and lactation through
a process mediated by immune cells [47,53], thus shaping the breast milk microbiome. This
provides a transient microbiota in the infant with great influence on the maturation of the
immune system in extrauterine life [3]. Therefore, the intestinal microbiome of women
during pregnancy and lactation could modulate the microbiota of human milk, suggesting
the importance of an adequate diet and nutritional status of the mother to achieve a healthy
microbiota that can subsequently colonize the breast milk that the infant receives.
In relation to maternal nutritional status, weight gain during gestation is important to
ensure fetal growth and development. Weight gains that exceed or fall below the established
recommendations have been associated with perinatal complications. In this study, it was
observed that breast milk samples from women with gestational weight gain above the IOM
recommendations [15] showed a tendency to lower alpha diversity: a finding that coincided
with that reported by Cabrera et al. [54], who identified that women with an obese BMI
during lactation and excessive weight gain during gestation tended to have a less diverse
bacterial community in their milk, with a higher relative abundance of Staphylococcus spp.,
and lower relative abundance of Bifidobacterium spp. Contrary to what was reported by
Lundgren et al. [10], when analyzing 155 breast milk samples, they observed greater alpha
diversity in the milk microbiota of women with higher gestational weight gain. Other
investigations have reported no differences in relation to maternal weight [55]. Our findings
highlight the importance of nutritional surveillance for the control of gestational weight
gain during pregnancy, not only because of its multiple implications for the health of the
Microorganisms 2023, 11, 1812 16 of 19

mother-child binomial [56] but also because of the impact it could have on the microbiota
and early colonization of the infant.
According to the results obtained from our study, it was found that the consumption
of simple carbohydrates during gestation and lactation was positively correlated with
Enterobacter spp., while the intake of dietary fiber during the first trimester of lactation
presented a negative correlation, which is important since this bacterial genus is character-
ized by opportunistic pathogenic species, which are associated with nosocomial diseases
and hospital infections [57]. On the other hand, the intake of simple carbohydrates during
lactation was negatively correlated with Bifidobacterium spp., an important bacterial genus
for infant health, which is considered a primary colonizer of the gastrointestinal tract of
the infant due to its ability to take advantage of the oligosaccharides in breast milk; its
reduction has been associated with the development of metabolic diseases [58].
The above is relevant, finding that 72% of lactating women participating in this study
exceeded the %AMDR [26] established in this country for simple carbohydrates, which
was represented mainly by the intake of “panela”(raw sugar cane cubes, which is a typical
product of some Colombian regions), sugar and sugary drinks. On the contrary, it is
noteworthy that the intake of fiber was very low, and 97% of women did not achieve
the recommendations.
Other relationships of interest have identified the consumption of folic acid during
lactation was positively correlated with Akkermansia spp.: a genus that is considered
a potential new generation probiotic with biotherapeutic actions for health in different
metabolic disorders and other health alterations associated with intestinal dysbiosis [59].
In this study, it was identified that the main food sources of folate consumed by lactating
women were cereals and fortified flours. In addition, some women reported continuing
the consumption of their folic acid supplementation; however, 87% presented a risk of
deficiency in the usual intake of this nutrient. Therefore, the consumption of food sources
of this micronutrient, including legumes and green leafy vegetables, as well as folic acid
supplementation in women at risk of deficiency, could be a dietary intervention strategy
that favors the presence of probiotic bacteria such as Akkermansia spp. in breast milk.
In this group of lactating women, a high prevalence in risk of deficiency in the usual
intake of micronutrients such as vitamin A, C, B6, B1, B3, zinc, and magnesium were
identified, and it was also found that the consumption of foods belonging to fats, fruits
and vegetables, meats, eggs, legumes, nuts, and seeds did not reach the recommendation
proposed in the Dietary Guidelines for lactating women in Colombia [60]. This could
negatively impact the microbial configuration of the mother and, therefore, that of the
newborn. In Colombia, health programs have focused on nutrition during pregnancy, but
the nutrition of lactating women has not been attended to, which has serious repercussions
on the characteristics of human milk, its richness, and bacterial diversity, which is essential
for the newborn.
Although this group of women enjoyed food and nutrition security at home, they
presented an inadequate intake of macro and micronutrients, which may be conditioned by
food choices that do not contribute to a diverse, healthy diet and favor the consumption of
risk nutrients such as simple carbohydrates. The results of this and other research make it
relevant to focus on lactating women because of the implications that their nutrition has on
the milk microbiota and maturation of the immune system in the first years of life.

5. Limitations of the Study

During gestation, the use of the frequency of food consumption generated an overesti-
mation of calcium intake. This is the first observational study conducted in this country,
and future trials and intervention studies are needed to validate our findings.

6. Conclusions
This study is the first in Colombia to explore the relationship between nutritional
status and feeding during gestation and lactation with the composition of breast milk
Microorganisms 2023, 11, 1812 17 of 19

microbiota. In this group, we observed that the diet of women could be related to genera
of interest for maternal and child health; we observed a negative correlation between
the lactation intake of simple carbohydrates and pregnancy intake with saturated fat and
the genus Bifidobacterium spp.; furthermore, a positive correlation was identified between
the lactation intake of folic acid and Akkermansia spp. These results contribute to new
knowledge in maternal and infant nutrition and favor the bacterial ecosystem through
interventions that contribute to healthy food choices and the feeding patterns of women
during the reproductive cycle.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/microorganisms11071812/s1. Table S1: concordance test
to assess reproducibility between 24 h recall and Food Consumption Frequency; Table S2: initial
and post-filter sequencing readings for each sample. Table obtained from QIIME2 software v2019.7;
Figure S2: Rarefaction curves. The horizontal axis represents the number of sequencing reads sampled
and the vertical axis the number of taxa reached to detect for each sample; Figure S1: Bacterial phyla
identified in breast milk microbiota.
Author Contributions: Conceptualization, D.C.L.-S.; Data curation, D.C.L.-S. and V.M.; Formal
analysis, D.C.L.-S. and V.M.; Funding acquisition, S.L.R.-M.; Investigation, D.C.L.-S. and L.B.P.;
Methodology, D.C.L.-S., V.M., N.C.G., L.B.P., O.I.M.-C. and S.L.R.-M.; Project administration, D.C.L.-S.
and S.L.R.-M.; Resources, S.L.R.-M.; Software, D.C.L.-S., V.M. and N.C.G.; Supervision, S.L.R.-M.;
Validation, D.C.L.-S. and N.C.G.; Visualization, D.C.L.-S. and V.M.; Writing—original draft, D.C.L.-S.
and L.B.P.; Writing—review and editing, D.C.L.-S., V.M., O.I.M.-C., L.B.P. and S.L.R.-M. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the University of Antioquia through resources from the
Committee for the Development of Research -CODI- Call for Research Projects Regionalization
2021-Project 2021-42724 and the EXITO Colombia Foundation: Project 2021-42724.
Data Availability Statement: Not applicable.
Acknowledgments: To the participating health institutions: Hospital San Juan de Dios de Rionegro
and ESE Nuestra Señora de la Candelaria of the municipality of Guarne Antioquia, Colombia.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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