ARTICLE

https://doi.org/10.1038/s41467-019-13118-0 OPEN

Characterization of antibiotic resistance genes in

the species of the rumen microbiota
Yasmin Neves Vieira Sabino1, Mateus Ferreira Santana1, Linda Boniface Oyama2, Fernanda Godoy Santos2,
Ana Júlia Silva Moreira1, Sharon Ann Huws2* & Hilário Cuquetto Mantovani 1*
1234567890():,;

Infections caused by multidrug resistant bacteria represent a therapeutic challenge both in

clinical settings and in livestock production, but the prevalence of antibiotic resistance genes
among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well
characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico
and confirm representative phenotypes in vitro. We find a high abundance of genes encoding
tetracycline resistance and evidence that the tet(W) gene is under positive selective pres-
sure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element
in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes
confirm the expression of the most abundant antibiotic resistance genes. Our data provide
insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and
reveal the potential role of mobile genetic elements in shaping the resistome of the rumen
microbiome, with implications for human and animal health.

1 Departamento de Microbiologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. 2 Institute for Global Food Security, School of Biological

Sciences, Queen’s University Belfast, Belfast, UK. *email: s.huws@qub.ac.uk; hcm6@ufv.br

NATURE COMMUNICATIONS | (2019)10:5252 | https://doi.org/10.1038/s41467-019-13118-0 | www.nature.com/naturecommunications 1

ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13118-0

W
ithout immediate action to tackle the current and ResFinder13, Resfams14, and ARG-ANNOT15 varied according to
escalating threat of antimicrobial resistance (AMR), it differences in specificity, sensitivity, and search method used by
is estimated that resistant infections may kill one each computational tool. ResFinder identified a total of 141
person every 3 s by the year 2050, raising the death toll worldwide acquired ARGs distributed in 72 rumen microbial genomes and
to 10 million annually1. This antibiotic-resistance crisis can be across 11 antibiotic classes (Table 1). ARG-ANNOT detected 754
linked to several issues, including the overuse of these compounds genes from 10 distinct antibiotic classes in 93 genomes of rumen
for medical and agricultural purposes, inappropriate antibiotic bacteria, while Resfams predicted 3148 sequences related to the
prescribing, and limited discovery/development of novel and resistance of 9 classes of antibiotics in 430 rumen microbial
effective antibiotics2. The widespread use of antibiotics in the genomes (Table 1; Supplementary Fig. 1). The most abundant
agricultural sector plays an underrepresented role in the AMR ARGs detected by all three bioinformatic tools were related with
context. Large amounts of antimicrobials are frequently used in resistance to beta-lactams (726 genes), glycopeptides (510), tet-
livestock to prevent diseases and promote animal growth3. More racycline (307), and aminoglycosides (193).
than 2 million kilograms of medically important antibiotics were Resfams detected the majority of the aminoglycosides and
sold in the USA for use in cattle in 2017, which corresponded to glycopeptides-resistance genes, while ARG-ANNOT identified
42% of the total used in food-producing animals, and ruminants the most beta-lactam-resistance genes in the rumen bacterial
have been recognized as a potential reservoir of antibiotic- genomes. Fluoroquinolone, fosfomycin, nitroimidazole, and
resistance genes (ARGs)4,5. trimethoprim were the antibiotic classes with the lowest number
The rumen ecosystem is colonized by a complex and genetically of ARGs identified using ResFinder, Resfams, and ARG-ANNOT.
diverse microbiota, in which dense populations of microbes often These putative ARGs were more prevalent among members of the
exist in close proximity within biofilms6. Therefore, mechanisms phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobac-
that lead to horizontal acquisition of novel genes could provide teria, but their abundances varied according to the bioinformatic
competitive advantage for resource competition and facilitate the tool used for ARG detection (Table 1; Supplementary Fig. 2).
exchange of genetic material between members of the rumen
microbiota and also with allochthonous species that occupy the
same site in the ruminant gastrointestinal tract (GIT)7–10. In addi- Phylogenetic distribution of ARGs. Although ARGs were widely
tion, feeding antimicrobials through ruminant diets can select for distributed across the genomes of ruminal bacteria (Fig. 1),
resistant organisms, potentially modifying the autochthonous resistance to specific antibiotic classes were more prevalent in
ruminal microbiota11. These findings corroborate with recent ana- some bacterial taxa, especially at the family and genus level.
lyses of the ovine rumen resistome, which identified resistances to
30 known antibiotics, including high abundance of genes for dap- Table 1 In silico detection of ARGs in ruminal microbial
tomycin and colistin resistance, two clinically relevant antibiotics5. genomes
Ruminants represent a major source of animal protein for
human consumption worldwide (through milk and meat pro-
General features ResFinder ARG- Resfams
duction), and cattle are raised in close proximity with humans, ANNOT
such as in rural areas, particularly in lower-middle-income
ARGs detected 141 754 3148
nations, where a great proportion of the livestock farms are small,
Genomes with ARGs 72 93 430
family-owned properties. Nonetheless, little is known about the ARGs by antibiotic class/
occurrence and distribution of ARGs among the bacterial species function
of the core rumen microbiome. Recently, hundreds of reference Aminoglycosides 10 26 157
genomes of cultured ruminal bacteria and archaea have been Beta-lactams 11 446 269
made available through the Hungate Project (http://www. Colistin 0 0 0
hungate1000.org.nz/), representing ~75% of the genus-level bac- Fosfomycin 2 2 0
terial and archaeal taxa present in the rumen12. We therefore Fluoroquinolones 4 4 2
hypothesized that genome mining of the Hungate genomic Fusidic acid 0 0 0
Glycopeptides 22 76 412
resources could reveal the distribution and genetic context of
MLS 22 44 44
ARGs within major ruminal species represented in the core Nitromidazole 3 0 0
rumen microbiome. Oxazolidinone 0 0 0
As such we analyzed 435 genomes of ruminal bacteria and Phenicols 3 10 19
archaea to search for ARGs using different computational Rifampicin 0 0 0
approaches. Identified ARGs were also evaluated for selective Sulfonamides 2 2 0
pressure, genetic organization, association with mobile genetic Tetracyclines 61 130 116
elements, as well as their expression in different rumen meta- Trimethoprim 1 14 0
transcriptome data sets. The resistant phenotype was confirmed for ABC efllux – – 1749
some cultured representative ruminal bacteria from the sequenced RND efllux – – 165
Othersa – – 215
Hungate1000 collection using in vitro testing and a novel inte-
ARG’s by taxonomic category
grative and conjugative element (ICE) associated with tetracycline Actinobacteria (n = 32) 7 11 164
resistance was identified in the genomes of rumen bacteria. Our Bacteroidetes (n = 50) 30 67 199
findings shed light on the distribution and frequency of ARGs Fibrobacteres (n = 2) 0 0 4
among the main species of bacteria colonizing the rumen, thereby Firmicutes (n = 313) 89 233 2502
improving our understanding of the mechanisms underlying Fusobacteria (n = 1) 0 0 2
antibiotic resistance within this microbial ecosystem. Proteobacteria (n = 22) 15 443 244
Spirochetes (n = 5) 0 0 27
Euryarchaeota (n = 10) 0 0 6
Results MLS Macrolides, Lincosamides, Streptogramins
Detection of ARGs in ruminal microbial genomes. The number aARGs that could not be ranked in any of the above antibiotic classes/functions; (−) these
ARGs are not included in the ResFinder and ARG-ANNOT data sets
and classes of ARGs detected in rumen microbial genomes using

2 NATURE COMMUNICATIONS | (2019)10:5252 | https://doi.org/10.1038/s41467-019-13118-0 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13118-0 ARTICLE

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Prevotellaceae bacterium KH2P17 Ruminococcus sp NK3A76

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Prevotella sp KH1P2
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Selenomonas ruminantium WCT3

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9236
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04 6 0
4

Fig. 1 Antibiotic-resistance distribution in ruminal microbes. The 16S rRNA gene sequences used to construct the tree were obtained from the
Hungate1000 collection and aligned using RDP Aligner. The phylogenetic tree was generated with the Maximum-Likelihood method using FastTree (1000
replicates), and visualized and annotated using iTOL. Black circles outside the tree represent the genomes, in which ARGs were identified from at least two
bioinformatics tools applied in this study

Members of the Proteobacteria phylum showed a high proportion rRNA gene phylogeny (Fig. 2). Majority of the genomes (69.2%)
of genomes harboring resistance genes, with ARGs being detected showed resistance to one antibiotic class, with a dominance of
in all genomes of the Enterobacteriaceae family. In the Bacter- tetracycline-resistance genes, which were distributed in bacterial
oidetes and Actinobacteria phyla, ARGs were concentrated in the genomes within the phyla Actinobacteria, Firmicutes, Proteobac-
genus Bacteroides and Bifidobacterium, respectively. In the Fir- teria, and Bacteroidetes. Phylogenetic analysis of the most
micutes phylum (which corresponded to the largest number of abundant tetracycline-resistance genes (tet(W), tet(Q), and tet
genomes analyzed in this study), resistance genes were detected (O)) revealed that tet(W) was highly conserved across different
across members of the Lachnospiraceae (Lachnosclostridium, taxa of ruminal bacteria, with a minimum sequence identity of
Clostridium, and Blautia) and Veillonellaceae (Selenomonas, 94.9 and 99% coverage in at least 28 ruminal bacterial genomes
Megamonas, and Megasphaera) families, as well as in all species of (Supplementary Figs. 3–5).
the genus Enterococcus and among strains of Staphylococcus Moreover, as shown in Fig. 2, metronidazole and fosfomycin
epidermidis. No antibiotic-resistance genes were detected in resistance were only identified in the genus Prevotella and in two
genomes belonging to the Spirochetes, Fibrobacteres, Fusobacteria, strains of Staphylococcus epidermidis, respectively. Staphylococcus
and Euryarchaeota phyla using Resfinder and ARG-ANNOT, but epidermidis together with Citrobacter sp. NLAE-zl-C269 were the
it should be noted that the number of genomes representing these only species harboring resistance genes to quinolones. Escherichia
taxa were much lower compared with the other phyla analyzed in coli PA-3 was the single ruminal genome showing the highest
this study. Although Resfams detected some potential ARGs in number of ARGs (n = 5), including tetracycline resistance,
these microbial taxa, the majority were ABC efflux pumps. The beta-lactam resistance, aminoglycoside resistance, trimethoprim
ABC efflux pumps represent one of the largest protein families in resistance, and sulfonamide resistance. Bacteroides ovatus NLAE-
microorganisms, contributing not only to reduce the intracellular zl-C500 was the only species in Bacteroidetes-possessing genes for
concentrations of toxic compounds but also to the influx of sulfonamide resistance. Beta-lactam-resistance genes were con-
substrates and other nutrients, and are not necessarily related to centrated in the family Enterobacteriaceae, in the genus
antibiotic resistance16. Bacteroides, and in two clades harboring the genera Selenomonas,
The distribution of the ARGs in ruminal microbial genomes Staphylococcus, Succiniclasticum, and Bacillus. Proteus mirabilis
was represented by the antibiotic class and according to the 16S NLAE-zl-G534 and Proteus mirabilis NLAE-zl-G285 shared the

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Tree scale: 0.01

0.987

0.998 0.89

1
0.937

0.976

0.984

0.997

0.938

1 1

0.999

0.875

0.739 0.997

0.9

0.987

0.912

0.989
1
1

0.996

0.864 0.961

0.927
0.985

0.999

0.866

0.94

0.989
0.985
0.993

0.964

0.883

0.973

0.959

0.999

0.918

0.984

1 0.912
0.876

0.995
0.976

0.888

0.965
0.986
1

1
1

0.999

0.986

0.853

0.977

0.947

Fig. 2 Distribution of ARGs in species of ruminal bacteria. The distribution of the resistance genes detected by at least two bioinformatic tools used in this
study is presented in front of the tree as filled colored boxes. The 16S rRNA gene sequences used to construct the tree were obtained from the
Hungate1000 collection and aligned using RDP Aligner. The tree was generated with the Maximum-Likelihood method using FastTree (1000 replicates)
and visualized and annotated using iTOL. Only bootstrap values >0.7 are shown

4 NATURE COMMUNICATIONS | (2019)10:5252 | https://doi.org/10.1038/s41467-019-13118-0 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13118-0 ARTICLE

a Blautia schinkii DSM 10518

5000 10,000 15,000 20,000 25,000

se j ns se e r al se ase e
So oJ ei rD
4 tW as 70 ato irD1 tion n pA pW lea rB
6
as aE
na Sp ot Pa Vi Te er a ul V iliza tei Re Dn n Vi er Tr
bi pr AT
f m g Tn c bi f
m ns Si
g re ob pr
o n u m ns
co ge ltr
a
al M do e co ltr
a
Re Ph
a hy it o
n E n R hy
et ip et
M
s cr M
an
Tr
Acessory genes Regulation

Mobilization Tetracycline resistance

Recombination Unknown function

b 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10,000 10,500 11,000 11,500 12,000 12,500 13,000 13,500 14,000 14,500 15,000 15,500 16,000 16,500 17,000 17,500 18,000 18,500 19,000 19,500 20,000 20,500 21,000

Blautia schinkii DSM10518.fasta

–2000 –1500 –1000 –500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10,000 10,500 11,000 11,500 12,000 12,500 13,000 13,500 14,000 14,500 15,000 15,500 16,000

Butyrivibrio hungatei DSM14810.fasta

–1000 –500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10,000 10,500 11,000 11,500 12,000 12,500 13,000 13,500 14,000 14,500 15,000 15,500 16,000

Lachnoclostridium aminophilum DSM10710.fasta

–3000 –2500 –2000 –1500 –1000 –500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10,000 10,500 11,000 11,500 12,000 12,500 13,000 13,500 14,000 14,500 15,000 15,500 16,000 16,500 17,000 17,500 18,000

Lachnoclostridium citroniae NLAE-zl-G70.fasta

–4500 –4000 –3500 –3000 –2500 –2000 –1500 –1000 –500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10,000 10,500

Lachnoclostridium lavalense NLAE-zl-G277.fasta

–3500 –3000 –2500 –2000 –1500 –1000 –500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10,000 10,500 11,000 11,500 12,000 12,500 13,000 13,500 14,000 14,500 15,000 15,500 16,000

Pseudobutyrivibrio sp. NOR37.fasta

–2500 –2000 –1500 –1000 –500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10,000 10,500 11,000 11,500 12,000 12,500 13,000 13,500 14,000 14,500 15,000 15,500 16,000

Pseudobutyrivibrio sp. OR37.fasta

Fig. 3 ICE_RbtetW_07 structure and conservation. a ICE_RbtetW_07 structure in Blautia schinkii DSM 10518. The genes of the ICE are represented by arrows
classified and colored in modules according to their function. The essential modules of the ICE machinery are represented in orange, blue, and pink, for
mobilization, recombination, and regulation genes, respectively. The ICE is located in scaffold 17 (nucleotide positions from 88079 to 111915) of the Blautia
schinkii DSM 10518 genome. b Alignment of the ICE_RbtetW_07 from ruminal bacterial genomes Blautia schinkii DSM 10518, Butyrivibrio hungatei DSM
14810, Lachnoclostridium aminophilum DSM 10710, Lachnoclostridium citroniae NLAE-zl-G70, Lachnoclostridium lavalense NLAE-zl-G277, Pseudobutyrivibrio sp.
NOR37, and Pseudobutyrivibrio sp. OR37. Lines connecting blocks with identical colors represent the aligned regions, and show synteny or gene
rearrangements. The tet(W) gene is represented in blue in the center of the figure

same pattern of resistance to tetracycline and phenicol, while involved in bacterial conjugation, such as relaxases, plasmid
Staphylococus epidermidis AG42 and Staphylococus epidermidis mobilization proteins (e.g., MobC), and conjugation proteins
NLAE-zl-G239 harbored highly homologous resistance genes for (e.g., TraE, TraG/TraD). In addition, genes enconding phage
beta-lactams, quinolones, and fosfomycin. proteins (e.g., PhiRv2) were detected in the flanking regions of the
tet(W) genes in ten ruminal microbial genomes (Supplementary
Data 1). Nonetheless, the scaffolds carrying the tet(W) genes
ARGs genetic context. The genetic locus of the tet(W) genes as lacked complete mobile elements showing the structural organi-
well as their flanking regions were analyzed to investigate their zation commonly found in plasmids, phages, and transposons.
mobility potential. For this, the 28 genomes harboring tet(W) A more detailed analysis of the sequences flanking the tet(W)
genes detected by ResFinder were evaluated in sequences of up to gene revealed that at least seven genomes contained the complete
4000 bp within the scaffold where tet(W) was located. Genes machinery required for a functional ICE, despite the fact that no
encoding a putative methyltransferase protein were detected such genetic elements were found when the bacterial genomes
adjacent to or located immediately upstream or downstrem of the were screened for using ICEberg17. The genetic organization of
tet(W) gene in 18 genomes. From these, the gene encoding the the ICEs identified in the genomes of ruminal bacteria, as
protein under the accession number D1PK82 [https://www. exemplified for Blautia schinkii DSM 10518 (Fig. 3a), contained
uniprot.org/uniprot/D1PK82] was the most prevalent, and putative genes encoding for conjugation proteins (e.g., TraE),
showed 98.9–100% sequence identity across the genomes of secretion proteins (e.g., VirD4), proteins controlling the cellular
ruminal bacteria. In seven of these genomes, a gene encoding the cycle (e.g., RepA, DnaI, σ70), and recombinases, in addition to
Maff-2 protein, which is known to be involved in bacterial other accessory proteins commonly found associated with these
resistance to tetracycline, was also found flanking the tet(W) gene. mobile elements (e.g., ATPases, methyltransferases, transposons,
Genes encoding transposon proteins (e.g., TnpW, TnpV, Tnp, and phage-related proteins). The hypothesis that a novel ICE
TnpX, and ISPsy9) were identified flanking the tet(W) gene in 10 (named here as ICE_RbtetW_07) could be mediating tetracycline
ruminal microbial genomes, and 22 out of the 28 genomes har- resistance in the genomes of rumen bacteria was reinforced by the
boring tet(W) genes also had genes encoding proteins potentially presence of conserved elements (reversed organization in three

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ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13118-0

L N B a 5

10,037 2
8000
6000
5000 1
4000
3000
2500 0
0 50 100 150 200 250 300 350 400 450 500 550 600 650
2000
b 5
1500

1000 4

800 3

dN/dS
600
2

400
1

200 0
0 50 100 150 200 250 300 350 400 450

Fig. 4 PCR detection of the ICE_RbtetW_07 in Blautia schinkii DSM 10518.
The expected amplicon size of the region from the virD4 gene to the tet(W)
gene was 4794 pb. L: 1 kb HyperLadder, N: negative sample, and B: Blautia
schinkii DSM 10518
genomes) in the ICE carrying the tet(W) genes on ruminal
bacterial chromosomes (Fig. 3b). Moreover, phylogenetic analyses
indicated that the ICE_RbtetW_07 had low sequence homology to
other integrative conjugative elements previously reported
(Supplementary Fig. 6).
Detection of the ICE_RbtetW_07 in B. schinkii DSM 10518. To
confirm the presence of the ICE carrying the tet(W) genes in rumen
bacteria, Blautia schinkii DSM 10518 was grown in the batch cul-
ture, and a fragment of 4794 bp covering the region from the virD4
gene to the tet(W) gene was PCR amplified from the genomic DNA
(Fig. 4). The amplicon was subjected to shotgun sequencing per-
formed on the MiSeq with 300 cycles using the next-Flex Illumina
workflow, and the assembled reads confirmed the genetic organi-
zation of the ICE (type IV secretion system gene adjacent to the
tetracycline-resistance gene) predicted in the Blautia schinkii DSM
10518 genome (accession number PRJNA223460, [https://www.
ncbi.nlm.nih.gov/bioproject/223460]) with 100% of nucleotide
identity and 99.7% of coverage (Supplementary Data 2).
Selection pressure analysis of tet resistance genes. Because the
tetracycline-resistance genes tet(W), tet(Q), and tet(O) were
highly abundant and broadly distributed in the genomes of
ruminal bacteria, we investigated if these genes are evolvable and
if genetic variants are being selected for in the genomes of
ruminal bacteria. For this purpose, the number of non-
synonymous per synonymous substitutions (dN/dS ratio) was
calculated to determine the codon substitution rate, which could
indicate the evolution of the gene in ruminal microbial genomes.
Our analysis confirmed the presence of positive selection pres-
sure in the tet(W) gene, with a dN/dS ratio > 1 for 36 out of the
639 amino acid residues in the protein (Fig. 5a). Sites showing
positive selection were mainly clustered in three distinct regions
of the TetW protein, located between amino acids positions
c 5
4
3
2
1
0
0 50 100 150 200 250 300 350 400 450 500 550 600 650
Site
Fig. 5 Selection pressure analysis in tetracycline-resistance genes. The
dN/dS ratio was calculated for the tet(W) (a), tet(Q) (b), and tet(O) genes
(c) using JCoDA. The lines in each panel represent dN/dS scores by site.
The phylogenetic tree generated by JCoDA for dN/dS analysis was
reconstructed using the aligned protein sequences of the ARGs and
Maximum-Likelihood approach with 100 replicates. Source data are
provided as a Source Data file
141–265, 310–341, and 600–639, with an average dN/dS ratio per
site of 1.9, 2.6, and 3.1, respectively. For the tet(Q) and tet(O)
genes, our analysis indicated 25 and 17 amino acid sites with
positive selection pressure, respectively (Fig. 5b, c). Nonetheless,
the overall dN/dS ratio calculated for these genes was <1, and the
likelihood ratio test data (LRT) for the substitution models did
not present statistically significant difference at 95% confidence
level, indicating absence of positive selection for tet(Q) and tet
(O) genes. To confirm that the tet(W) gene is under positive
selection, neutrality tests were applied in these ARG sequences.
The results of Tajima’s D (D = −1.65) and Fu and Li test (D* =
−2.22. p < 0.05; F* = −2.39, p < 0.05) corroborate with our
previous findings.
Confirmation of resistance phenotypes. To validate the resis-
tance phenotype that was computationally predicted in the gen-
omes of ruminal bacteria, 26 pure cultures matching the genomes

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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13118-0
Bacteria BLA AMG
Acetitomaculum ruminis DSM 5522
Bifidobacterium adolescentis DSM 20087
Bifidobacterium boum DSM 20432
Bifidobacterium merycicum DSM 6492
Bifidobacterium pseudolongum DSM 20092
Bifidobacterium thermophilum DSM 20212
Blautia schinkii DSM 10518
Butyrivibrio fibrisolvens DSM 3071
Butyrivibrio proteoclasticum B316
Butyrivibrio sp. M55
Lachnoclostridium aerotolerans DSM 5434
Clostridium intestinale DSM 6191
Clostridium lundense DSM 17049
Lachnoclostridium aminophilum DSM 10710
Lachnoclostridium polysaccharolyticum DSM 1081
Lachnospira multiparus D15d
Lactobacillus ruminis DSM 20403
Megasphaera elsdenii T81
Mitsuokella jalalundinii DSM 13811
Olsenella umbonata DSM 22619
Prauserella rugosa DSM 43194
Proteiniclasticum ruminis DSM 24773
Ruminococcus flavefaciens FD-1
Selenomonas ruminantium DSM 2872
Sharpea azabuenensis DSM 18934
Sharpea azabuenensis DSM 20406
Fig. 6 Concordance between antibiotic resistance detected in silico and in vitro. Twenty six bacteria were analyzed both in silico and in vitro in this study.
Colors represent the approach used to predict the ARG or to detect the resistance phenotype of each bacteria. BLA (beta-lactam); AMG (aminoglycoside);
TET (tetracycline); VAN (vancomycin); CHL (Chloramphenicol); MAC (macrolide); CLD (clindamycin)
analyzed in silico were selected for further in vitro characteriza-
tion. Our in silico analysis predicted 57 resistance determinants in
the ruminal bacteria that were selected for in vitro testing.
Minimum inhibitory concentration (MIC values) were deter-
mined using the Epsilometer test (E-test) method18, and 18
resistance phenotypes were confirmed in vitro (Supplementary
Table 1). From this, eight ARGs (~44.0%) were detected/con-
firmed both in silico and in vitro by all approaches used in this
study (Supplementary Fig. 7). ARG-ANNOT predicted 14 ARGs
in the 26 cultures of ruminal bacteria tested in vitro, and 10
resistance phenotypes (71.4%) were confirmed in the E-test
assays. ResFinder and Resfams predicted 14 and 53 ARGs in the
bacterial cultures, and resistance to 9 (64.3%) and 17 (32.1%) of
these antibiotics were confirmed phenotypically (Supplementary
Fig. 7). Therefore, resistance phenotypes predicted by ResFinder
and ARG-ANNOT agreed, for the most part, with the in vitro
results, while Resfams predicted several resistance mechanisms
that could not be confirmed by other computational or in vitro
methodologies used in this study.
In addition, tetracycline resistance was detected simultaneously
by at least three approaches in 69% of the genomes and bacterial
cultures, providing further evidence that tetracycline resistance is
widespread in the rumen ecosystem. Moreover, some resistance
phenotypes were confirmed by our in vitro assays even when
the presence of resistance determinants were predicted by only one
bioinformatic tool. This was observed for vancomycin resistance in
Clostridium and Lachnoclostridium strains and in Lactobacillus
ruminis DSM 20403, for beta-lactams in Mitsuokella jalalundinii
DSM 13811 and Selenomonas ruminantium DSM 2872, for
aminoglycosides in Bifidobacterium pseudolongum DSM 20092
and for macrolides in Prauserella rugosa DSM 43194 (Fig. 6).
Expression of ARGs in metatranscriptomes. To confirm if the
ARGs detected in ruminal microbial genomes are expressed in the
rumen ecosystem, we aligned the genes conferring resistance to
the five most abundant antibiotic classes (aminoglycoside, beta-
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ARTICLE
TET VAN CHL MAC CLD
ResFinder
ARG-ANNOT
In vitro
Resfams
lactam, macrolide, tetracycline, and vancomycin) detected in
rumen bacterial genomes to 15 rumen metranscriptome data sets
of dairy and beef cattle and sheep. At least one resistance gene
from each antibiotic class was expressed in one or more meta-
transcriptomes (Fig. 7). In general, tetracycline-resistance genes
presented the highest level of expression, mainly the tet(O), tet
(Q), tet(W), and tet(37) gene. Moreover, individual genes varied
in their expression levels between different data sets, being more
prevalent in sheep and beef cattle. Some of the ARGs identified in
the ruminal microbial genomes, such as strA, cepA, ermB, tet(B),
and vanC, among others, were not expressed in any of the
metatranscriptome data sets investigated in this study (Fig. 7).
Discussion
The hypothesis that livestock animals represent a reservoir of
ARGs is not new, and previous studies have demonstrated that
bacteria isolated from fecal samples of monogastrics and rumi-
nants harbor genetic elements that can confer resistance to
clinically used antibiotics19. However, less attention has been
given to the occurrence of ARGs in the rumen ecosystem and
their potential to be transferred to commensal and pathogenic
bacteria. Microorganisms colonizing the rumen also disseminate
to the environment through animal saliva during rumination and
due to the flow of rumen microbial biomass to the omasum,
abomasum and to the distal parts of the GIT, being released
through fecal discharge into the soil20.
In this work, we investigated the occurrence and distribution of
ARGs in hundreds of ruminal microbial genomes made available
through the Hungate1000 project12. Our results indicate that
ARGs are widely distributed among ruminal bacteria, with genes
conferring resistance to tetracycline being frequently detected in
the genomes of several species. The regions flanking the
tetracycline-resistance genes showed a conserved pattern in an
apparently novel integrative and conjugative element (ICE),
suggesting dissemination of antibiotic resistance through hor-
izontal gene transfer in the rumen microbiome. In addition, we
7
ARTICLE
SRR8050517
SRR8050516
SRR8050514
SRR3257011
SRR3256999
SRR5805554
SRR5805529
SRR3169847
SRR3169851
SRR6201277
SRR6201276
SRR1138697
SRR594654
SRR873462
SRR873454
aadE
blaACl -1 rpkm
mef(A)
tet (32)
tet (40)
tet (J)
tet (O)
tet (Q)
tet (W)
tet (37)
vanR-C
vanR-D
Beef cattle Dairy cattle Sheep
Fig. 7 Expression levels (RPKM) of ARGs in ruminal metatranscriptomes.
Beef and dairy cattle and sheep data sets were analyzed, and only ARGs
that were expressed in at least one metatranscriptome are represented.
Source data are provided as a Source Data file
show evidence of positive selective pressure on the tet(W) gene,
which may contribute to the selection of genetic variants of this
gene, conferring ribosome protection against tetracycline anti-
biotics in ruminal bacteria.
Tetracyclines are broad-spectrum antibiotics that inhibit pro-
tein synthesis in susceptible bacteria21 and have been used in
human and veterinary medicine to fight bacterial infections and
to promote the growth of food-producing animals, improving
feed efficiency and animal performance22. The growth-promoting
activity of tetracyclines in livestock was first reported in 1949
when feeds supplemented with dried Streptomyces aureofaciens
mash were administrated to poultry23. However, the long-term
use of subtherapeutic doses of tetracyclines has been associated
with increased levels of bacterial resistance in the GIT of food-
producing animals24, which led countries such as the USA and
members of the European Union to ban the use of tetracyclines as
growth promoters since 197525. The prevalence of tetracycline-
resistance genes among ruminal bacteria has a potential negative
impact in livestock health given that tetracyclines account for up
to 24% of the antibiotics used to treat respiratory, uterine, or
locomotion diseases in Europe26.
In our study, tet(W), tet(Q), and tet(O) were the main
tetracycline-resistance genes identified by ResFinder, but tet(W)
was the most abundant gene, being distributed in 28 genomes of
ruminal bacteria with high similarity of nucleotide sequences.
Since ResFinder is an online platform that uses whole-genome
sequencing data to identify acquired AMR genes in bacteria13, the
tet(W) gene was selected for further analysis with an aim to
investigate if it could be horizontally transferred among ruminal
bacteria.
Our analyses suggest that the tet(W) gene of several species of
ruminal bacteria are located on an ICE with a unique modular
pattern, not previously reported for tetracycline resistance
(ICE_RbtetW_07). The organization of the ICE structure is char-
acterized by a core region containing essential modules for con-
jugation, recombination, and regulation27. The conjugation
machinery often contains relaxases and secretion systems to med-
iate the transfer of DNA between donor and recipient cells. The
recombination module usually contains genes encoding integrases,
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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13118-0
excisionases, and recombinases, while genes encoding proteins
involved in DNA transcription are found in the regulation module.
Moreover, several accessory genes, including methyltransferases,
transposons, phages, and plasmid related proteins, in addition to
the ARGs, can be present in an ICE structure27. At least one
essential gene from each module and several accessory genes were
detected in the flanking region of the tet(W) gene in seven ruminal
0
microbial genomes analyzed in this study, suggesting that ICEs
0.607
could be mediating the horizontal transfer of tetracycline-resistance
1.214 genes between ruminal bacteria. Some essential genes of integrative
1.82 and conjugative elements were also found in other genomes of
2.427 rumen bacteria harboring the tet(W) gene, but the genetic structure
3.034
of these elements appeared incomplete. The structure of ICE_Rb-
3.641
tetW_07 was conserved in the genomes of ruminal bacteria, and the
4.248
4.854
presence of the tet(W) gene on the ICE was confirmed in the
5.461
genome of Blautia schinkii DSM 10518 by PCR amplification and
6.068 shotgun sequencing. ICEs are known to be frequently transferred
between distant bacterial taxa28, and our analysis suggest that these
genetic elements might have great mobility in the rumen ecosystem.
If this is indeed the case, there is a potential threat of these genetic
elements to be transferred to bacterial pathogens in the GIT of
ruminants and into the environment.
Some groups of microorganisms, such as the Enterobacter-
iaceae, are frequently associated with the horizontal transfer of
ARGs29,30. The enrichment of ARGs appears to be a common
feature in the mobile resistome of Proteobacteria from both the
animal and the human gut microbiome31. This may be asso-
ciated with the frequent use of antibiotics targeting Enter-
obacteriaceae to control infections in the respiratory, urinary,
and GIT of humans and livestock, thus selecting for resistant
strains5. Our in silico results demonstrated that members of the
Proteobacteria phylum that inhabit the rumen can harbor
multiple ARGs, as exemplified by the genome of E. coli PA-3, a
ruminal isolate that contains five genes conferring resistance to
four distinct antibiotics. Although generic Escherichia coli
populations are often not so abundant in the rumen (<106 cells/
ml), cattle are considered a natural reservoir for pathogenic
strains of E. coli32,33. In addition, management and nutritional
practices, such as feeding the animals a high-grain diet, can
cause significant increases in ruminal and fecal populations of E.
coli and influence the abundance and diversity of ARGs in the
ruminal resistome33,34.
Furthermore, a complete operon for vancomycin resistance
(vanC) was found in the genomes of Enterococcus casseliflavus
and in Enterococcus gallinarum SKF1, which makes these species
intrinsically resistant to this antibiotic35. Vancomycin is one of
the last-option antibiotic used to treat infections caused by Gram-
positive bacteria36, but little is known about vancomycin resis-
tance in ruminal bacteria. Our analyses predicted resistance to
vancomycin in Clostridium, Lachnoclostridium, and Blautia, and
in vitro experiments confirmed these phenotypes in Blautia
schinkii DSM 10518, Lachnoclostridium aerotolerans DSM 5434,
Clostridium intestinale DSM 6191, and Clostridium lundense
DSM 17049. However, our transcriptomic analysis indicated
that the expression of vancomycin-resistance genes in the
rumen microbiome is low. In addition, vancomycin has limited
use for therapeutic purposes in animals, and vancomycin analogs
have been restricted as growth promoters in livestock36.
Overall, our in vitro assays confirmed 31.6% of the resistance
phenotypes predicted by computational approaches. Nonetheless,
64.3% and 71.4% of the resistance phenotypes based on the ARGs
predicted by ResFinder and ARG-ANNOT, respectively, were
confirmed in vitro. These results show concordance between
these different approaches, despite the fact that some predicted
ARGs might not be functional in vitro. In addition, our tran-
scriptomic analyses showed higher expression of tetracycline-
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-13118-0
resistance genes and low expression of other classes or ARGs
(such as beta-lactams and aminoglycosides) in the rumen data
sets, which also agreed with the resistance phenotype observed
in vitro for the pure cultures of ruminal bacteria. Therefore, the
results presented here, based on genomic, transcriptomic and
phenotypic data, increase the confidence that ARGs are dis-
tributed among different species of ruminal bacteria, and that
these genes are expressed by members of the microbial commu-
nity in vivo. Nonetheless, additional studies will be needed to
evaluate the effects of animal ageing, production systems, and
nutritional practices, including antibiotic feeding, on the expres-
sion, and potential transit of the ARGs detected in this study
across the GI tract of cattle.
Taken together, our results provide insights that characterize
the antibiotic resistance in species of ruminal bacteria. Our
findings demonstrate that ARGs, in particular the genes for tet-
racycline resistance, are prevalent among ruminal bacteria and
subjected to positive selection pressure. Moreover, analysis of the
flanking regions of the tet(W) genes provided evidence that this
gene can be disseminated horizontally through ICE transfer.
Given the fact that the rumen ecosystem also harbor a highly
dense and diverse microbiota that maintain complex ecological
associations, and the capacity of microorganisms to exchange
DNA through direct (conjugation) and indirect mechanisms
(transformation, transduction), the GIT of ruminants should be
considered as a relevant source of antibiotic-resistance genes. The
evidence of a new ICE (ICE_RbtetW_07) carrying the tet(W) gene
emphasizes this idea and presents a risk for both animal and
human health, due to the capacity of these genetic elements to
disseminate into the environment. This hypothesis is also sup-
ported by the observation of non-phylogenetically related bacteria
harboring the same resistance genes, and the expression of the
same ARGs in ruminal metatranscriptomes data sets from beef
and dairy cattle and sheep. In addition, our results highlight the
importance of using multiple computational tools to search for
ARGs in genomic data and demonstrate the relevance to validate
these results in vitro and/or in vivo.
Methods
Collection of genomic data and identification of ARGs. The sequence files of 435
ruminal microbial genomes (425 bacteria and 10 archaea), from the Hungate1000
project, were downloaded in FASTA format from the NCBI (National Center for
Biotechnology Information, http://www.ncbi.nlm.nih.gov/genome) and the JGI
(Joint Genome Institute, http://genome.jgi.doe.gov) websites (Supplementary
Data 3). Putative ARGs in the bacterial and archaeal genomes were predicted using
three bioinformatics tools (ResFinder v2.1, Resfams v1.2, and ARG-ANNOT Nt
V3, see below) that vary in sensitivity and specificity and apply distinct databases
and search methods to detect putative ARGs in nucleotide sequences.
We initially prospected the bacterial and archaeal genomes using the ResFinder
v2.1 database (https://cge.cbs.dtu.dk/services/ResFinder/)13 against all antibiotic
classes available for analysis of acquired ARGs, which included aminoglycosides,
beta-lactams, colistin, fluoroquinolones, fosfomycin, fusidic acid, glycopeptides,
MLS (macrolides, lincosamides, and streptogramins), nitroimidazole,
oxazolidinone, phenicols, rifampicin, sulfonamides, tetracyclines, and
trimethoprim. For homology-based screening, the minimum gene identity and
sequence length were set to 70 and 60%, respectively, in relation to the reference-
resistance gene.
The software Resfams v1.2 was used to search for conserved structural domains
of antibiotic-resistance function in protein sequences of ruminal bacteria14. The
protein sequences of the analyzed genomes were downloaded in the FASTA format
from the NCBI or JGI websites and when these sequences were not found, the
online interface Prodigal v1.2 (http://compbio.ornl.gov/prodigal/server.html) was
used to convert nucleotide to protein sequences using the Standard Bacteria/
Archaea translation table code.
Genomes of ruminal microorganisms were also analyzed using the ARG-
ANNOT database (http://en.mediterranee-infection.com/article.php?
laref=283&titre=arg-annot-)15. Sequences of the ARGs in the ARG-ANNOT
database (ARG-ANNOT Nt V3) were used for similarity analysis against the
ruminal microbial genomes using BLASTn37 on the Galaxy platform38. The
alignment parameters were minimum sequence identity of 70%, sequence length
cutoff of 60%, and E-value < 10–6.
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ARTICLE
Phylogenetic analysis and distribution of ARGs. To evaluate if the ARGs
grouped according to the evolutionary history of the microbial species, phyloge-
netic trees were reconstructed using the 16S rRNA gene sequences from the gen-
omes analyzed in this study. The 16S rRNA gene sequences were obtained from the
Hungate1000 collection12. Next, all the sequences were organized in a single txt file
and aligned using the RDP Release 11.5 aligner in the Ribosomal Database Project
website (RDP, https://rdp.cme.msu.edu/)39. FastTree v2.1 (http://www.
microbesonline.org/fasttree/)40 was used to infer Approximately-Maximum-
Likelihood phylogenetic trees using default settings. The generated output file
(.tree) was visualized and annotated on the Interactive Tree of Life (iTOL) interface
v4 (https://iTOL.embl.de/)41.
To evaluate sequence conservation of the ARGs that were identified in silico as
described above, the sequences of the most frequent genes conferring resistance
were extracted from the ruminal microbial genomes, and aligned as described
below. To extract the sequences of the resistance genes, all genomes were annotated
by Prokka v1.1242 using the Galaxy platform (minimum contig size of 200 and E-
value < 10–6). To obtain the location of the resistance genes in the bacterial
genomes, the ResFinder v2.1 database was aligned against the annotated genomes
using the BLASTn tool on the Galaxy platform. Minimum query coverage and
sequence identity were 60 and 70%, respectively, with a threshold E-value < 10–6.
The sequence of the resistance genes identified in the previous alignment were
extracted using the SAMtools software v1.9 (http://SAMtools.sourceforge.net/)43,
and the most abundant ARGs were aligned using MUSCLE version 3.8.3144. The
phylogenetic reconstruction was based on the Maximum-Likelihood method using
FastTree v2.1, and iTOL v4 graphical interface was used to represent the
phylogenetic trees, as described above.
Genetic context of ARGs. To assess potential mechanisms of ARG mobility,
the genetic elements flanking the resistance genes in a scaffold were investigated
using the software Artemis45. When these flanking elements were annotated as
hypothetical protein, sequence function was predicted on the UNIPROT website
(http://www.uniprot.org/)46, using BLASTn, UniProtKB as the target database and
default settings. Putative mobile genetic elements flanking the resistance genes were
searched using different computational tools. ISfinder (https://www-is.biotoul.fr/)47
was used to screen for transposons and/or integrons, and phage sequences were
confirmed using PHASTER (http://phaster.ca/)48. The genomes that harbored
proteins with predicted plasmid functions near the resistance genes were further
analyzed against the Enterobacteriaceae and Gram-positive database of Plas-
midFinder 2.0 (https://cge.cbs.dtu.dk/services/PlasmidFinder/)49, using a threshold
for minimum sequence identity of 70% and minimum coverage of 60%. Finally,
ICEberg 2.0 (http://db-mml.sjtu.edu.cn/ICEberg/)17 was used to identify ARGs
carried by integrative and conjugative elements (ICE). Scaffolds harboring selected
ARGs were used as query sequences (FASTA format) in the WU-BLAST2 search
tool in ICEberg, using BLASTn as the search program and ICE nucleotide sequence
as the sequence database. All the other parameters were kept as default settings.
Conservation and in vitro confirmation of a novel ICE. To evaluate the genetic
conservation of the integrative and conjugative elements identified in the genomes
of ruminal bacteria, the nucleotide sequences of these ICEs were aligned using
progressiveMAUVE50. In addition, a phylogenetic tree was reconstructed in MEGA
X software using Muscle to compare the sequences of the novel ICE with other
ICEs available in the ICEberg database. Phylogenetic reconstruction was performed
using the Maximum-Likelihood method with 100 replicates.
To confirm the presence of a novel ICE in the genome of ruminal bacteria, PCR
amplifications were performed using Blautia schinkii DSM 10518. Primers were
designed to amplify a 4794 pb DNA fragment that included the region from gene
virD4 to gene tet(W). Briefly, the bacteria was grown in anaerobic media at 39 °C
for 72 h. Genomic DNA was extracted using the Qiagen DNA extraction kit
(Manchester, UK). PCR amplification of the region of interest was carried out in
25 µl volumes as follows: 12.5 µl 2x MyTaq HS Red Mix, 1 µl each of 20 µM primers
drawn in this study (RbtetW07_F: ATGAAAAAGCAGCTTGACATCAAAAAGC
and RbtetW07_R: TTACATTACCTTCTGAAACATATGGCGC), 8 µl of nuclease-
free water, and 2.5 µl of 50 ng template DNA. The cycling conditions were as
follows: initial denaturation step at 95 °C for 60 s, 35 cycles of denaturation at 95 °C
for 15 s, annealing at 58 °C for 15 s, and extension at 72 °C for 3 min, and a final
extension step at 72 °C for 5 min. The size and integrity of PCR products were
visualized and assessed by agarose gel electrophoresis using a 1% (w/v) agarose in
TAE buffer (40 mM Tris, pH 8.0, 20 mM acetic acid, 1 mM EDTA, BioRad Ltd.,
Hemel Hempstead, UK). The PCR product was subjected to shotgun sequencing
performed on the MiSeq with 300 cycles using the next-Flex Illumina workflow at
the Queen’s University of Belfast Genomics Core Technology Unit (Belfast, UK) to
confirm the presence of the predicted integrative and conjugative element. Quality
filtering of the reads was performed using Trimmomatic software v0.2751, and
sequences were assembled using SPAdes 3.10.152. The sequence was aligned to the
ICE coding sequence in the genome of Blautia schinkii DSM 10518, using Clustal
Omega53.
Selective pressure on identified ARGs. Sequences of the most prevalent ARGs
detected in ruminal microbial genomes were selected to evaluate if these genes are
9
ARTICLE
under selection pressure. Selective pressure on the ARGs was estimated by the
number of non-synonymous and synonymous substitutions (dN/dS) using the
software JCoDA54. JCoDA uses ClustalW to align the nucleotide sequences and
Phylogenetic Analysis using Maximum Likelihood (PALM) to calculate the dN/dS
ratio. Analyses were performed using nucleotide sequences in the FASTA format as
input sequences and the universal (default) genetic translation code. The alignment
option was set to ClustalW, and phylogenetic trees were reconstructed by the
Maximum-Likelihood method with 100 replicates using protein sequences in the
Phylip-sequential format. The dN/dS was calculated by site (amino acid) using the
Bayes Empirical Bayes (BEB) model, which takes into account the uncertainties of
model parameters, avoiding the generation of false positives54. The selective
pressure on the antibiotic-resistance genes were confirmed by two neutrality tests,
the Tajima test (performed in MEGAX55) and Fu and Li test (DnaSP v656).
In vitro analysis of the predicted resistance phenotypes. To evaluate if the
ARGs detected in the ruminal microbial genomes conferred the predicted resis-
tance phenotype in vivo, 26 cultures of ruminal bacteria that had their genomes
analyzed for the presence of ARGs were tested in vitro to confirm these pheno-
types. Cultures stored at −80 °C were activated twice in brain heart infusion (BHI)
broth at 39 °C for 24 h under anaerobic conditions before each experiment. For the
antibiotic susceptibility test, the optical density (OD600nm) of the cultures was
adjusted to obtain a cell count of ~108 CFU ml−1 (equivalent to 0.5 McFarland
scale). Subsequently, cultures were spread on the surface of solid BHI media using
swabs. E-test strips (Biomérieux, Basingstoke, UK; Fannin, Dublin, Ireland) of the
antibiotics to be tested were distributed on the surface of the inoculated medium,
and the plates were incubated anaerobically at 39 °C for another 24 h. Only anti-
biotics to which resistance was predicted in silico were evaluated in vitro using pure
cultures of ruminal bacteria. The minimum inhibitory concentration (MIC) of each
antibiotic was determined at the intersection of the inhibition zone with the E-test
strips57. All experiments were performed with three biological replicates. Reference
values from the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) breakpoint table were used to interpret the MIC results58. In the case of
a few antibiotics where breakpoint values were not available for the tested species,
MIC interpretations were based on breakpoint values published for the most clo-
sely related organism.
Metatranscriptomic analyses. The metatranscriptome data sets were downloaded
in the FASTQ format from the NCBI Sequence Read Archive SRA (https://www.
ncbi.nlm.nih.gov/sra)59. Fifteen ruminal data sets representing cattle from distinct
production systems (dairy and beef) and from sheep were selected for the analysis
(Supplementary Table 2). FastQC software v0.11.5 (http://www.bioinformatics.
babraham.ac.uk/projects/fastqc) was initially applied for quality control checks on
raw sequence data using a Phred quality score of 20 to filter high-quality bases.
However, because most reads were discarded using this criteria, gene expression
was evaluated using raw sequence data.
In this study, the most abundant group of ARGs identified in the ruminal
microbial genomes was selected to verify their expression in the
metatranscriptomic data sets. These genes were predicted to encode resistance to
aminoglycosides (aadE, ant(6)-Ia, aph(3’)-III, strA, strB), beta-lactams (cepA,
blaACl−1, blaSED1, blaDHA-2, blaZ), macrolides (ermB, erm(G), mef(A), Isa(A),
IsaC), tetracycline (tet(32), tet(40), tet(A), tetA(P), tet(B), tetB(P), tet(D), tet(J), tet
(M), tet(O), tet(Q), tet(W), tet(37), and vancomycin (vanC, vanD, vanH-D, vanR-
C, vanR-D, vanS-C, vanS-D, vanT-C, vanX-D, vanXY-C, vanY-D, vanZ-F). The
Bowtie2-build tool was used to index the DNA sequences of the resistance genes,
while Bowtie260 was employed to align these sequences to the metatranscriptomic
data sets. The expression level of each gene was calculated by the number of
uniquely mapped reads per kilobase in a gene per million mappable reads
(RPKM)61 using a cutoff value of 0.362.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article.
Data availability
The data that support the findings of this study are included in the paper and/or
its Supplementary Information files. Data underlying Figs. 5, 7 and Supplementary
Figs. 1, 2, and 7 are provided as a Source Data file.
Received: 5 January 2019; Accepted: 21 October 2019;
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Acknowledgements
This work has been supported by the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES; Brasília, Brazil), Fundação de Amparo a Pesquisa do Estado de
Minas Gerais (FAPEMIG; Belo Horizonte, Brazil), and the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq; Brasília, Brazil). We are also grateful
for RCUK Newton Institutional Link Funding (172629373).
Author contributions
Y.N.V.S., H.C.M., and S.A.H. conceived the project. Y.N.V.S. and A.J.S.M. completed the
laboratory work under the supervision of H.C.M. and S.A.H. Y.N.V.S., M.F.S., and F.G.S.
analyzed the data and generated figures. S.A.H. provided the anaerobic cultures analyzed
in the study. L.B.O. assisted Y.N.V.S. with the in vitro experiments. L.B.O., M.F.S.,
and S.A.H. provided valuable ideas into the project from the time of conception. Y.N.V.S.
and H.C.M. wrote the paper with imput from all co-authors.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41467-
019-13118-0.
Correspondence and requests for materials should be addressed to S.A.H. or H.C.M.
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