J. Dairy Sci.
105:4804–4817
https://doi.org/10.3168/jds.2021-21325
© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Changes of antibiotic resistance genes and gut
microbiota after the ingestion of goat milk
Yufang Liu and Fuxin Zhang*
College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710119, China
ABSTRACT
Antibiotic resistance genes, as newly emerging con-
taminants, have become a serious challenge to public
health through the food chain. The gut of humans and
animals is an important reservoir for the development
and dissemination of antibiotic resistance genes because
of the great abundance and diversity of intestinal micro-
biota. In the present study, we evaluated the influence
of goat milk on the diversity and abundance of antibi-
otic resistance genes and gut microbial communities,
especially pathogenic bacteria. Male mice were used, 12
for each of the 2 groups: a control group that received
sterile distilled water and a treated group that received
goat milk, and gut microbiota and antibiotic resistance
genes were compared in these groups using metage-
nomic analysis. The results revealed that ingestion of
goat milk decreased the diversity and abundance of
antibiotic resistance genes in the mice gut. The relative
abundance of fluoroquinolone, peptide, macrolide, and
β-lactam resistance genes in the total microbial genes
significantly decreased after the intervention. Goat
milk intake also significantly reduced the abundance of
pathogenic bacteria, such as Clostridium bolteae, Clos-
tridium symbiosum, Helicobacter cinaedi, and Helico-
bacter bilis. Therefore, goat milk intake might decrease
the transfer potential of antibiotic resistance gene to
pathogenic bacteria in the gut. In addition, bacteria
with multiple resistance mechanisms accounted for ap-
proximately 4.5% of total microbial communities in the
control group, whereas it was not detectable in the goat
milk group, indicating the total inhibition by goat milk
intake. This study highlights the influence of goat milk
on antibiotic resistome and microbial communities in
the gut, and provides a new insight into the function of
goat milk for further study.
Key words: antibiotic resistant gene, metagenomic
analysis, gut microbiota, pathogenic bacteria
Received September 22, 2021.
Accepted January 28, 2022.
*Corresponding author: fuxinzh@​snnu​.edu​.cn
4804
INTRODUCTION
Goat milk is an important part of human nutrition
because of its high nutritional values, hypoallergenicity,
and easy digestibility (Feng et al., 2019). Goat milk has
been demonstrated to be an excellent source of protein,
calcium, potassium, phosphorus, niacin, pantothenic
acid, riboflavin, thiamin, and vitamin A in the human
diet (Willett, 2003). Consumption of goat milk also
shows several health benefits, including improvements
in gastrointestinal disorders, growth rates, Fe and Cu
absorption, bone density, and blood levels of Ca, vita-
min A, riboflavin, and niacin (Stergiadis et al., 2019).
Apart from that, goat milk shares many essential
characteristics with human breast milk and, thus, has
been reported as one of the most important surrogates
for breast milk (Tannock et al., 2013; Clark and Mora
García, 2017).
The gut is a complex environment inhabited by nu-
merous microorganisms that form a greatly diverse and
active ecological community (Khurana et al., 2020).
These intestinal microbes are of great importance to
several functions of the host, such as defense against
invasion by foreign organisms, maintaining the normal
physiology and homeostasis, and development of the im-
mune system (Sommer et al., 2017; Zheng et al., 2020).
Related studies on intestinal microbiota of individuals
have demonstrated that these commensal microbiota
in the gut can serve as reservoirs of antibiotic resis-
tance genes due to their great diversity and abundance
(Achard et al., 2020). These resistance bacteria have
potential to spread and transfer antibiotic resistance
genes to pathogens via horizontal genes transfer, thus
helping them increase their duration of persistence
and possibilities of survival in the gut without selec-
tive antibiotic pressure (Ghosh et al., 2013; Li et al.,
2021). This transfer paves the way for pathogens to
invade the gut of the host and aggravate the clinical
situations of infectious diseases (Juricova et al., 2021;
Langdon et al., 2021). Thus, antibiotic resistance genes
are generally regarded as an emerging genetic pollutant
and have become one of the most serious challenges to
public health (Ding et al., 2020; Li et al., 2020). The
 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA
resistant strategies include acquisition of related genes
facilitating the breakdown or efflux of the antibiotics,
mutations of antibiotic targets, and alternate pathways
to escape the attack of antibiotics (Wang et al., 2022).
In the last decades, studies on the selection and develop-
ment of antibiotic resistance genes have largely focused
on environmentally and clinically relevant bacteria (Jia
et al., 2021). The risks of antibiotic resistance genes
in the intestinal microbiota should be an important
point, given the likelihood that resistance genes may
be directly transferred to pathogenic bacteria in the
gut. Therefore, it is of great importance to improve
the existing knowledge regarding the distribution of
antibiotic resistance genes in the intestinal microbiota
and their alteration after dietary intervention.
Previous studies showed that goat milk could modu-
late the composition of microbial community in the gut,
and these alterations in the diversity and abundance
of some microbial genera could provide several health
benefits to the host (Tannock et al., 2013; Zhang et
al., 2018; Niyazbekova et al., 2020). In this study, we
hypothesized that ingestion of goat milk might eventu-
ally result in changes in the composition of antibiotic
resistance genes in the gut microbiota. Thus, the aim
of this work was (1) to compare the profiles of antibi-
otic resistance genes and microbial communities in the
intestinal microbiota with and without goat milk intake
using a metagenomic approach, and (2) to explore the
relationships of goat milk, antibiotic resistance genes,
and gut microbiota. This work provided new insight
into the function of goat milk for further study.
MATERIALS AND METHODS
Collection of Milk Samples
Goat milk samples were collected from 30 Saanen
dairy goats in the farm of Northwest Agriculture and
Forestry University (Xianyang, China). All the dairy
goats were fed with a mixture of maize silage, maize
grain, alfalfa hay, wheat bran, and mineral premix, as
reported by Shi et al. (2017). The process for collection
of milk samples, animal experiments, and extraction
of metagenomic DNA from the fecal samples has been
previously described in detail (Liu et al., 2021). Briefly,
all dairy goats in this work were healthy and fed with
standard feeding materials. Before collecting the goat
milk manually, the udder surface of each dairy goats
was rinsed with sterile distilled water and sterilized
with 70% ethanol. After discarding the first 3 milk
drops from each dairy goat, goat milk was collected
and placed into sterile plastic tubes, numbered, and
transported to the laboratory immediately in an ice
box.
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4805
Animal Experiments
Specific-pathogen-free C57BL/6J mice (male; 12
wk old) were purchased from Vital River Laboratory
Animal Technology Co. Ltd. All mice were reared in
a standard animal facility at 20°C to 24°C with 12 h
light/dark cycles, and fed a standard chow diet and
sterilized water ad libitum. After 1 wk of acclimatiza-
tion, these mice were randomly divided into 2 groups:
goat milk and control group, which were intragastri-
cally administered with goat milk and sterile distilled
water. Each group consisted of 12 mice that were di-
vided into 3 cages to account for cage effects. Goat
milk was pasteurized by heating at 65°C for 30 min
before use. All mice in the goat milk group received
10 mL of goat milk per kg of BW daily for 4 wk and
those in the control group received the same volume of
sterile distilled water. The mice in different cages were
numbered differently (G1–G3 and C1–C3). At the end
of 28 d, fecal samples (250 mg) from each cage were col-
lected separately, frozen in liquid nitrogen, and stored
at −80°C before use. This work was approved by the
Scientific Ethics Committee of Shaanxi Normal Uni-
versity and all procedures were performed according
to the National Institutes of Health Guide for the Care
and Use of Laboratory Animals (National Institutes of
Health Publication No. 85–23, revised 1985).
DNA Extraction
Metagenomic DNA in the fecal samples were ex-
tracted using Qiagen DNA Mini-kit (Qiagen) according
to the manufacturer’s protocol. All samples were dis-
solved in PCR water and checked by gel electrophoresis
in 0.8% agarose at the voltage of 100 V for 40 min.
The concentration and purity of the metagenomic DNA
were assessed by NanoDrop 2000 spectrophotometer
(Thermo Fisher Scientific). The extracted DNA was
further diluted to 1 ng/μL in sterile distilled water as
the template for the PCR experiment. Diluted samples
were stored at −20°C until further use.
16S rRNA Gene Sequencing
To characterize microbial communities, the V3–V4
hypervariable regions of the bacterial 16S genes were
amplified by using PCR with the universal forward
primer 338F (5′-ACTCCTRCGGGAGGCAGCAG-3′)
and the reverse primer 806R (5′-GGACTACHVGGGT-
WTCTAAT-3′). A set of 6-base barcodes were added
to the primers for sorting of samples from sequencing
outcomes. Each PCR reaction mixture contained about
10 ng of template DNA and 0.2 μL of each primer in
15 μL of PCR Master Mix (Thermo Fisher Scientific).
 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA
Thermal cycling was done in triplicate with an initial
denaturation at 94°C for 3 min, followed by 30 cycles
of denaturation at 94°C for 30 s, annealing at 45°C for
30 s, and elongation at 72°C for 30 s, and terminated
by a final extension for 5 min at 72°C. The amplified
PCR products were purified by Qiagen Gel Extraction
Kit and assessed by gel electrophoresis in 2% agarose
for 40 min at a voltage of 80 V. After purification and
quantification, PCR products were subjected to DNA
sequencing by using the Illumina PE150.
Metagenomic Sequencing
Approximately 5 μg of DNA for each sample were sent
to Novogene Co. Ltd. for the library construction with
an insert size of 350 bp, as described previously (Zhao
et al., 2018). Subsequently, Illumina high-throughput
sequencing was performed on the Hiseq 4000 platform
using a 100-bp paired-end strategy. Approximately
6 Gb of metagenomic data was generated from each
sample, resulting in a total of over 210 Gb data output
from all samples. All the raw data were filtered by a
self-written script to remove the reads with a size less
than 100 bp. The 100-bp paired-end reads were merged
employing a self-written script to screen for 10- to 50-
bp overlap reads and to assemble them into 150- to
190-bp Illumina tags. In the next step, the number of
Illumina tags was standardized to 1 × 107 in each data
set for downstream bioinformatic analysis.
Bioinformatic Analysis
Microbial Community Analysis. All the data
analysis of the 16S genes were performed in Mothur
v. 1.35.1. Sequences were de-multiplexed and quality-
filtered using the Quantitative Insights into Microbial
Ecology (QIIME v1.8.0) to trim chimeric sequences. In
the next step, the normalization of the clean sequences
was processed by extracting randomly clean sequences
from each sample to compare with other samples at the
same sequence depth. Then, the normalized sequences
were clustered into operational taxonomic units (OTU)
with a threshold identity of 97%. The taxonomic clas-
sification of each sequence was conducted using the
Ribosomal Database Project classifier with a boot-
strap threshold of 80%. The phylogenetic relationships
among the representative sequences of the OTU were
analyzed using the Core Set database in Greengenes
(http:​/​/​greengenes​.lbl​.gov/​).
Antibiotic Resistance Genes Analysis. A widely
accepted antibiotic resistance gene database was down-
loaded from the comprehensive antibiotic resistance
database (CARD; McArthur et al., 2013; Alcock et al.,
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4806
2020). The redundant sequences from the downloaded
database were trimmed using a self-written script. The
metagenomic iTag from each sample were searched in
the nonredundant CARD using BLASTP with an E-
value <1 × 10−5. The antibiotic resistance genes were
classified into 19 antibiotic resistance gene types (e.g.,
tetracycline resistance gene) and 121 subtypes (e.g.,
tetQ).
Statistical Analysis
The mean and significant differences for variability of
antibiotic resistance gene type and pathogenic bacteria
were calculated by using Excel 2019 (Microsoft Office
2019, Microsoft Corporation). Pearson’s correlation and
other tests were conducted using SPSS v.21 software
(IBM Corp.). A heatmap was constructed to evaluate
the correlations of abundance of antibiotic resistance
genes and microbial communities at the genus level us-
ing Matlab 7.0 (MathWorks).
RESULT
Comparison of Antibiotic Resistance Genes Between
Goat Milk Group and Control Group
In total, 121 antibiotic resistance genes were detected
from the gene catalog of 314,655 gut microbial genes.
Analysis of similarity (ANOSIM) between the goat
milk group and control group showed that there were
greater differences in composition of antibiotic resis-
tance genes between 2 groups than among fecal samples
from the same group (r = 0.815, P = 0.1; Figure 1A).
This result was further confirmed by the multiple re-
sponse permutation procedure analysis, which showed
that differences between 2 groups were higher than the
differences within each group (A = 0.132, P = 0.07).
The total number of antibiotic resistance genes sig-
nificantly decreased from 100.33 ± 0.89 (data expressed
as mean ± SD) in the control group to 62.67 ± 1.56 in
the goat milk group (P < 0.01, Figure 1B). The shared
and unique antibiotic resistance genes among 2 groups
were shown in Figure 1C. Out of the 121 antibiotic
resistance genes detected in all samples, a total of 67
genes were shared by the goat milk group and control
group. Compared with the control group, 41 resistance
genes disappeared after goat milk intake, whereas 13
new resistance genes appeared. Thus, consumption of
goat milk could significantly reduce the diversity of
antibiotic resistance genes in the mouse gut microbiota.
The relative abundance of antibiotic resistance
genes decreased from 0.0814% in the control group to
0.0762% in the goat milk group, although the differ-
 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA 4807

Figure 1. Differences in diversity and abundance of antibiotic resistance genes between goat milk group (G) and control group (C). (A)
Similarity analysis (ANOSIM) of the difference in structure of antibiotic resistance genes of intestinal microbiota in 2 groups. (B) The total
number of antibiotic resistance genes in 2 groups. (C) Venn diagram of number of antibiotic resistance genes in 2 groups. (D) Relative abundance
of antibiotic resistance genes in 2 groups. (E) Relative abundance and distribution of antibiotic resistance genes in 6 samples. The circle diagram
was divided into 2 parts, antibiotic resistance gene information on the left and the sample information on the right. Inner ring: different colors
represent different antibiotic resistance genes and samples; the left scale represents the sum of relative abundance of antibiotic resistance gene
in all samples; the right scale shows the relative abundance of an antibiotic resistance gene in each sample. The left side of outer ring represents
the relative percentage of each antibiotic resistance gene in each sample and the right side represents the relative percentage of each antibiotic
resistance gene in a sample. Error bars indicate SD. **P < 0.01. ARG = antibiotic resistance genes.

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 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA
Figure 2. Relative abundance of antibiotic resistance gene types in the goat milk group and control group. Error bars indicate SD. MLS =
macrolide-lincosamide-streptogramin. *P < 0.05.
ence was not statistically significant (P > 0.05, Fig-
ure 1D). The tetQ and adeF genes were predominant
in all samples, accounting for 63.77% and 68.53% of
the total antibiotic resistance genes in the goat milk
group and control group, respectively. The abundance
of adeF genes were significantly reduced in the goat
milk group by 37.10% versus the control group (P <
0.05), whereas no significant difference was observed in
tetQ genes (P > 0.05). The antibiotic resistance genes
that significantly decreased mainly belonged to mdtB,
mdtF, arnA, adeF, and evgS, and the majority of the
increased antibiotic resistance genes were members of
the tet family (Figure 1E).
Differences in Antibiotic Resistance Gene Types
The 121 antibiotic resistance gene sequences were
classified into 19 different resistance gene types, and
the most abundant gene types were tetracycline, ami-
noglycoside, multidrug, aminocoumarin, fluoroquino-
lone, macrolide-lincosamide-streptogramin, peptide,
macrolide, β-lactam, and vancomycin resistance genes,
accounting for 98.5% of the total antibiotic resistance
gene abundances (Figure 2). The differences between
the relative abundance of these gene types in the 2
groups were calculated by Student’s t-test. Among
these 10 gene types, the abundance of 8 gene types
(tetracycline, multidrug, aminocoumarin, fluoroquino-
lone, macrolide-lincosamide-streptogramin, peptide,
macrolide, β-lactam) decreased, of which differences of
Journal of Dairy Science Vol. 105 No. 6, 2022
4808
fluoroquinolone, peptide, macrolide, and β-lactam were
statistically significant (P < 0.05). However, higher
abundances of aminoglycoside and vancomycin resis-
tance genes were observed in the goat milk group.
Compositional Differences
of the Microbial Community
To compare the microbial communities in the goat
milk group and control group, we performed 16S rRNA
gene sequencing for each sample. After the quality con-
trol and denoising removal, 381,385 high-quality reads
were generated from the 16S rRNA V3–V4 regions,
ranging from 6,056 to 6,854 reads with average reads of
6,397. All these reads were categorized into 3,299 OTU,
which were used for downstream analysis.
The result showed that at the phylum level, more
than 90 bacterial, archaeal, and eukaryotic phyla were
detected in all the samples. Among them, the phyla
Bacteroidetes, Firmicutes, Verrucomicrobia, Proteo-
bacteria, and Deferribacteres were present at a level
of above 0.1% of all bacteria; Bacteroidetes was the
predominant phylum in both groups, representing more
than 60% of all bacteria present (Figure 3A). However,
no significant differences in the abundance of intestinal
bacteria were observed among these 2 groups at the
phylum level.
At the genus level, 832 bacterial genera were found in
the mice gut, including Bacteroides, Prevotella, Akker-
mansia, Parabacteroides, Clostridium, Lachnoclostridi-
 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA 4809

Figure 3. Relative abundance of gut microbial communities in the goat milk group (G) and control group (C) at phylum level (A) and genus
level (B). (C) Linear discriminant analysis (LDA) effect size results on the intestinal microbiota of the goat milk group and control group of
mice. Representative bacteria in both groups at different taxonomic levels were presented (p: phylum, c: class, o: order, f: family, g: genus, and
s: species).

Journal of Dairy Science Vol. 105 No. 6, 2022

 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA
um, Hungatella, and Anaerotruncus (in order from high
to low abundance). Bacteroides was the predominant
genus, accounting for 15.0% and 16.5% of all bacteria
in the gut microbiota of both groups (Figure 3B). The
genera Prevotella, Clostridium, and Anaerotruncus were
more abundance in the goat milk group than that in the
control group. In contrast, Bacteroides, Akkermansia,
Parabacteroides, Lachnoclostridium, Hungatella, and
Coprobacillus were more abundant in the control group.
To further address taxonomic differences in the intes-
tinal microbiota, the abundant microbial communities
were assigned by linear discriminant analysis effect size
with linear discriminant analysis score greater than 4.0.
Representative bacteria at different taxonomic levels
(from phylum to species) in the intestinal microbiota
of goat milk group and control group were presented
in Figure 3C. Species Lachnospiraceae bacterium
A4, Prevotella sp. CAG485, Bacteroidales bacterium
5246, Anaerotruncus sp. G32012, and Mucispirillum
schaedleri were enriched in the gut of the goat milk
group. Hungatella hathewayi, Akkermansia muciniphila,
Lachnoclostridium bolteae, Bacteroides caecimuris, and
Helicobacter magdeburgensis were the key species in
the control group. Notably, several pathogenic bacteria
were detected in the control group as well, including
Helicobacter cinaedi, Bacteroides fragilis, Clostridium
clostridioforme, and Clostridium bolteae.
Comparison of Pathogenic Bacteria
As shown in Figure 4A, pathogenic bacteria ac-
counted for 1.08 to 1.32% of total intestinal bacteria in
the goat milk group and 1.79 to 1.82% in the control
group, respectively. A total of 28 pathogenic bacteria
were found in both groups. Clostridium spp. were the
dominant species, followed by Bacteroides fragilis, Heli-
cobacter typhlonius, Helicobacter cinaedi, Clostridioides
difficile, and Helicobacter bilis (Figure 4B). The abun-
dances of Clostridium bolteae, Clostridium symbiosum,
Helicobacter cinaedi, and Helicobacter bilis in the con-
trol group were 1.52, 1.93, 5.72, and 3.41 times higher
than those in the goat milk group, respectively (P <
0.05). Only one pathogenic bacterium, Helicobacter
typhlonius, significantly increased after consumption of
goat milk (P < 0.01).
Phylogenetic Origins of Antibiotic Resistance Genes
To compare the microbial origin of antibiotic resis-
tance genes with total microbial genes in the gut, all
antibiotic resistance genes and total gut microbial genes
were classified into different taxa with the support of
the Resistance Gene Identifier (RGI). In goat milk
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4810
group, different assignments of antibiotic resistance
genes and total microbial genes at the phylum level
were observed: 30% versus 61%, 14% versus 16%, 13%
versus 2%, and 2% versus 6% for Bacteroidetes, Fir-
micutes, Proteobacteria, and Verrucomicrobia (Figure
5A). In the control group, the distribution of antibiotic
resistance genes and total microbial genes at the phy-
lum level was 40% and 2% for Proteobacteria, 20% and
63% for Bacteroidetes, 9% and 15% for Firmicutes, and
2% and 8% for Verrucomicrobia, respectively (Figure
5B). This inconsistency suggests that compared with
other genes in the intestinal microbiota, antibiotic re-
sistance genes in both groups were more prone to exist
in Proteobacteria but less prone to occur in Bacteroide-
tes, which was consistent with the previous studies (Hu
et al., 2013; Li et al., 2020). The phylum Proteobacteria
was the predominant origin of antibiotic resistance
genes in the control group, and after ingestion of goat
milk, the relative abundance of antibiotic resistance
genes in this phylum was significantly reduced by 27%
(P < 0.05).
Correlation of Antibiotic Resistance Genes
With Microbial Communities
According to the previous study, it was speculated
that microbial communities could be the potential hosts
of antibiotic resistance genes if these genes and co-
existing microbial communities presented significantly
similar abundance tendencies between different samples
(P < 0.01; r > 0.6; Li et al., 2015a). In this work, cor-
relation between antibiotic resistance genes and the top
20 genera was investigated using heatmap analysis to
obtain the detailed information of antibiotic resistance
gene hosts. As shown in Figure 6, a significant cor-
relation was observed between the abundance of mtrD
and Ruminococcus, indicating that Ruminococcus was
the potential host of mtrD. Likewise, Hungatella and
Enterobacter were the possible host of evgS, and Para-
prevotella was the potential host of adeB and tet32. In
addition, there was no significant correlation between
the genus Helicobacter and antibiotic resistance genes.
Antibiotic Resistance Mechanism Categories
According to the CARD database, all the resistance
bacteria in this study were classified into 7 primary
categories based on their resistance mechanism: an-
tibiotic efflux, antibiotic target protection, antibiotic
target alteration, antibiotic inactivation, and 3 multiple
resistance mechanisms (antibiotic target alteration
and antibiotic efflux, antibiotic target alteration and
antibiotic efflux and reduced permeability to antibi-
 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA 4811

otic, and antibiotic efflux and reduced permeability DISCUSSION

to antibiotic). As shown in Figure 7, bacteria with
resistance mechanism of antibiotic efflux, antibiotic
target alteration, and antibiotic target protection were
present at levels of more than 10% of total microbial
communities; antibiotic efflux was the predominant
mechanism category, representing 69.2% and 65.5% of
all resistance mechanisms in the control group and goat
milk group, respectively. It was worth noting that bac-
teria with multiple resistance mechanisms accounted
for approximately 4.5% of total microbial communities
in the control group, whereas it was not detectable in
the goat milk group, indicating the total inhibition by
goat milk intake.
In this work, we compared the composition of mi-
crobial community and the distribution of antibiotic
resistance genes in the intestinal microbiota with and
without goat milk intake and gained insight into
whether there is an interaction between goat milk, gut
microbiota, and antibiotic resistance genes. The results
showed that ingestion of goat milk led to a decrease
in the abundance and diversity of antibiotic resistance
genes in the intestinal microbiota of mice. The resis-
tance genes for fluoroquinolone, peptide, macrolide, and
β-lactam were not detectable in the goat milk group,
indicating the total inhibition of antibiotic resistance

Figure 4. (A) Relative abundance of pathogenic bacteria goat milk group (G) and control group (C). (B) Relative abundance of pathogenic
bacteria species in the goat milk group (G) and control group (C).

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 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA 4812

Figure 5. Comparison of the distribution of antibiotic resistance genes (inner ring) and total microbial genes (outer ring) at the phylum level
in the goat milk group (A) and control group (B).

gene expression by goat milk intake. A previous study resistance genes and their carriers, and alleviation of
reported that dietary intervention, which consisted metabolic syndrome in obese children (Wu et al., 2016).
of whole grains, traditional Chinese medicinal foods, According to another previous study, the reduction in
and prebiotics, resulted in the diminution of antibiotic richness and diversity of the gut resistome might be a

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 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA 4813

Figure 6. Correlation between the top 20 antibiotic resistance genes and bacteria at the genus level. The figure shows the heatmap where
samples were clustered according to their composition. The r-value is shown in different colors. The P-value is indicated as *, **, and ***, which
indicate the correlation is significant at the 0.05, 0.01, and 0.001 level, respectively.

result of inhibitory response of their hosts to dietary
intervention (Tan et al., 2021). Hence, it is important
to assign the antibiotic resistance genes to their micro-
bial origin. In this study, it was found that compared
with other gut genes, antibiotic resistance genes in both
groups were more prone to exist in Proteobacteria but
less prone to occur in Bacteroidetes, which was in accor-
dance with previous studies (Hu et al., 2013). Related
studies also reported that some specific antibiotic resis-
tance genes, such as fluoroquinolone resistance genes,
β-lactamase resistance genes, glycopeptide resistance
genes, macrolide resistance genes, and multiple drug
resistance genes, were most frequently correlated with
Proteobacteria (Durso et al., 2012). The relative abun-
dance of antibiotic resistance genes in Proteobacteria
was significantly decreased after ingestion of goat milk,
indicating that altered composition of Proteobacteria
might be one explanation for antibiotic resistance gene
reduction.
Furthermore, correlation analysis between the top 20
genera and antibiotic resistance genes was investigated
to obtain the detailed information of antibiotic resis-
tance gene hosts. It was speculated that nonrandom co-
occurrence patterns between antibiotic resistance genes
and microbial communities could indicate the possible
information on antibiotic resistance gene hosts, if the
abundance of these resistance genes and microbial taxa
presented significantly similar tendencies between dif-
ferent samples (P < 0.01; r > 0.6). In other words,
the corresponding similar abundance tendencies could
be attributed to some specific microbial taxa carrying
certain antibiotic resistance genes (Li et al., 2015b).
In the present study, heatmap analysis revealed that
15 bacterial genera were speculated as the hosts of

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 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA 4814

Figure 7. Percentage of bacteria with different resistance mechanisms in the goat milk group (A) and control group (B).

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 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA
10 antibiotic resistance genes in the mouse gut. Some
hosts of antibiotic resistance genes have been verified in
previous studies. For instance, the genus Eubacterium
was reported to be the potential host of tet32 by a
previous study (Lancaster et al., 2005). In recent years,
correlation analysis was widely applied to explore the
potential hosts of antibiotic resistance genes in various
samples, including landfill leachate, fecal samples, and
an urban river (Wu et al., 2016; Zhao et al., 2018; Jia
et al., 2021). Therefore, correlation analysis is a reason-
able tool to provide us new insights into the potential
hosts of antibiotic resistance genes, but further valida-
tion was required by a culture-based method, function
metagenomic approach, or metagenomics assembly
(Munir et al., 2011; Mullany, 2014).
According to related studies, gut microbiota contains
many bacteria that possess several antibiotic resistance
genes, and these genes can transfer in the gut from one
bacterium to another distantly related bacterium, espe-
cially pathogenic bacteria (Salyers et al., 2004; McInnes
et al., 2020). These pathogenic species confer both an-
tibiotic resistance and pathogenicity, and lead to more
treatment failures and higher mortality, which poses
an increasing threat to public health. The results in
this work indicated that abundance of both antibiotic
resistance genes and pathogens in the goat milk group
was lower than that in the control group, although dif-
ferences in the abundance of antibiotic resistance gene
were not statistically significant. Therefore, goat milk
intake might decrease the transfer potential of antibi-
otic resistance genes to pathogens in the gut.
In addition, the dominant category of antibiotic re-
sistance mechanism detected in both groups was the
antibiotic efflux pump, and the relative abundance of
bacteria with multiple resistance mechanisms decreased
from approximately 4.5% in the control group to zero
after goat milk intake. Efflux pumps are transport
proteins that exclude or pump not only intracellular
antibiotics but also toxic substrates and heavy met-
als from the bacterial cells to the external environ-
ment (Shi et al., 2021). Therefore, the antibiotic efflux
pump was the most efficient mechanism of fighting
widely against various environmental stresses, leading
to the prevalence of antibiotic resistance genes with
the efflux pump mechanism (Zhang et al., 2015). As
fewer studies metagenomically characterized antibiotic
resistance genes in intestinal microbiota after dietary
intervention, it was hard to speculate which food com-
position can inhibit certain antibiotic resistance types.
One reasonable explanation was that intestinal bacteria
carrying antibiotic resistance genes with different resis-
tance mechanisms responded dissimilarly in an altered
environment, such as different content of short-chain
Journal of Dairy Science Vol. 105 No. 6, 2022
4815
fatty acids and nutrient substances in the gut. Further
studies on the relationship between the alteration of
antibiotic resistance genes and intestinal microbiota
target dietary intervention would be of interest.
Using the metagenomics approach, we first provided
an in-depth insight into the changes of antibiotic re-
sistance genes in the gut microbiota after goat milk
ingestion. However, little attention in this study was
currently paid to related mobile genetic elements (e.g.,
plasmids and integrons) in the gut microbiota, which
play a vital role in the horizontal transfer of antibiotic
resistance genes. To determine if antibiotic resistance
genes spread into pathogenic bacteria in the gut, in-
depth genome-centered metagenomic analysis should
be performed in further research to evaluate the in-
teraction of antibiotic resistance genes, mobile genetic
elements, and pathogenic bacteria in the gut, and to
explore the underlying mechanisms on the horizontal
transfer of antibiotic resistance genes.
CONCLUSIONS
Our results revealed that ingestion of goat milk de-
creased the diversity and abundance of antibiotic resis-
tance genes in the gut of mice. The relative abundance
of fluoroquinolone, peptide, macrolide, and β-lactam
resistance genes significantly decreased after the inter-
vention (P < 0.05). Ingestion of goat milk also signifi-
cantly reduced the abundance of pathogenic bacteria,
such as Clostridium bolteae, Clostridium symbiosum,
Helicobacter cinaedi, and Helicobacter bilis (P < 0.05).
Therefore, goat milk might decrease the transfer poten-
tial of antibiotic resistance genes to pathogenic bacteria
in the gut. In addition, bacteria with multiple resis-
tance mechanisms accounted for approximately 4.5%
of total microbial communities in control group, but
were not detectable in goat milk group, indicating total
inhibition by goat milk intake. These results provide
important implications on the function of goat milk for
further study.
ACKNOWLEDGMENTS
This study was supported by the Key Industry Inno-
vation Chain of Shaanxi Province (2020ZDLNY02-08,
China), National Natural Science Foundation of China
(31801566), and Innovation Support Plan of Shaanxi
Province (2021TD-31, China). Author contributions
were as follows: Yufang Liu: conceptualization, writ-
ing, and investigation; and Fuxin Zhang: supervision,
reviewing, and funding acquisition. The authors have
not stated any conflicts of interest.
 Liu and Zhang: ANTIBIOTIC RESISTANCE GENES AND GUT MICROBIOTA
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ORCIDS
Fuxin Zhang https:​/​/​orcid​.org/​0000​-0001​-7826​-3028