Human Microbiome Journal 10 (2018) 43–52

Contents lists available at ScienceDirect

Human Microbiome Journal

journal homepage: www.elsevier.com/locate/humic

Human microbiomes and antibiotic resistance T

a a a,⁎
Sophie A Baron , Seydina M Diene , Jean-Marc Rolain
a
Aix-Marseille Univ., IRD, APHM, MEPHI, IHU Méditerranée Infection, Facultés de Médecine et de Pharmacie, 19-21 Boulevard Jean Moulin, 13385 Marseille Cedex 05,
France

A R T I C LE I N FO A B S T R A C T

Keywords: Human microbiomes are complex ecosystems involving bacteria, viruses, archaea or eukaryotes that are co-
Microbiota evolving in an environment subject to various selective pressures, such as antibiotic administration, diet and/or
Resistance genes lifestyle. In this sympatric lifestyle, competition is hard and the synthesis of antibiotic molecules and/or anti-
Sympatric life biotic resistance genes (ARGs) is one solution that was developed by the organisms to survive. This environment
Gene transfer
becomes a large source of ARGs for pathogenic bacteria, leading to the risk of infection due to multidrug re-
Metagenomic
Culture
sistant bacteria. Culture and metagenomics are two complementary methods developed to study these micro-
biomes in order to better understand the type of bacteria and ARGs present in the human body, as well as the
factors that modulate the abundance and variety of these ARGs. The objective of this review was to identify
factors that influence the colonization and propagation of multidrug resistant bacteria and/or ARGs, and to
define resistance genes and multidrug resistant bacteria that have already been isolated from the human mi-
crobiota using culturomics and metagenomics techniques.

1. Introduction co-evolution and was developed by Van Valen [6]. Applied to antibiotic
resistance, this hypothesis implies that under antibiotic selective pres-
The microbiome was first defined by Joshua Lederberg as the eco- sure, such as antibiotic administration or multiple stress factors pro-
logical community of commensal, symbiotic, and pathogenic micro- duced in the gut by human immunity, bacteria will acquire mechanisms
organisms that literally share our body space [1]. For more than of resistance or virulence to survive. However, this theory could be also
10 years, an international project has been studying human micro- applied on interbacterial competition. Antibiotic resistance is ancient
biomes of several body sites, such as the gut, airways, skin, urine or [7], and this arms race started long time before the introduction of
genital microbiomes, particularly with two methods: metagenomics and antibiotic in human medicine [8]. Bacteria always fight in a competi-
culturomics [1]. This project has led to the discovery of new microbial tive environment to survive by acquiring resistance genes [7,8]. These
species and allowed identification of specific microbiotas and features genes are generally acquired through bacteriophages, plasmids or
of these different sites. Among them, antibiotic resistance genes (ARGs) transposons. Human microbiomes are complex ecosystems where
present in the microbiota have been studied [2]. The human body sites multiple organisms live that are continually in competition, leading to a
shelter a wide variety of bacteria, viruses, archaea, or eukaryota [3,4]. real “arms race”. This evolutionary theory is opposed to the Court
The balance between all these organisms is complex, and organisms are Jester theory [9], which supposes that a spontaneous event leads to the
developing defense mechanism such as antibiotic secretion or antibiotic evolution of a species. These two theories were previously opposed for a
resistance products synthesis to keep their advantage in the ecosystem. long time, but recently it has been proposed that they coexist and in-
These defense mechanisms contribute to the resilience of the normal terplay. As such, the “Alice’s living croquet” theory, also referring to the
microbiota and in fine to the host which can resist in an antibiotic- book Through the looking glass, has been proposed recently by Raoult
polluted world. These can be intrinsic to commensal bacteria or can be [10] to explain the impossibility of predicting the occurrence of events
acquired by transitional bacteria which need defenses in hostile areas in an environment where several living organisms are evolving. This
[2,5]. Five hypotheses can be presented to understand these host-bac- theory, first suggested by Gregory Bateson [10], supposed that in an
teria-gene relations. The Red Queen hypothesis or “the arms race hy- environment where several living organisms interact, it is impossible to
pothesis” (from Through the looking glass by Lewis Carroll) is based on predict the behavior of one organism compared to another, hence the

⁎
Corresponding author at: IRD, APHM, MEPHI, IHU Méditerranée Infection, Faculté de Médecine et de Pharmacie, Aix-Marseille Université, 19-21 Boulevard Jean
Moulin, 13385 Marseille Cedex 05, France.
E-mail address: jean-marc.rolain@univ-amu.fr (J.-M. Rolain).

https://doi.org/10.1016/j.humic.2018.08.005

Available online 15 November 2018

2452-2317/ © 2018 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
S.A. Baron et al. Human Microbiome Journal 10 (2018) 43–52

RESISTANCE GENE ORIGIN FACTORS INFLUENCING

RESISTOME

Resident resistome Antibiotics

Resident microbiota Food
Virome Country/Travel
Antibiotic use in
livestock
Transitory resistome
Urban or rural
Transitory microbiota lifestyle
Transitory virome

Bacteriophage
Free DNA

Horizontal
conjugative
transfer Porine

Chromosomal
insertion

Traduction of
resistant genes

Fig. 1. Different types of antibiotic resistance gene exchange in the microbiota and factors influencing the resistome.

possibility for a clone to spread independently of any selection pressure; are good settings for all these theories: (i) they contain multiple or-
this clone itself could carry antibiotic resistant genes [10]. The un- ganisms [3], (ii) they are good reservoirs for resistance genes, and (iii)
predictability of this behavior can also be illustrated with the pendulum their environment predisposes to gene transfer, sometimes leading to
(multiple oscillator) metaphor [11]. As such, the behavior of a bacterial the emergence and spread of a specific clone carrying genes of re-
clone might be variable, but independent variabilities also occurs for sistance and/or virulence or the gene itself. The objective of this review
plasmids, transposons or genes [12]. This situation can be schematized was, first, to identify factors that influence colonization and propaga-
as a double -or multiple- oscillator (a second pendulum attached to the tion of multidrug resistant bacteria and/or ARGs and, second, to define
end of the first); if we increase the number of evolutionary units (e.g. genes of resistance in multidrug resistant bacteria that have already
transposons, integrons, genes), we have a multi-oscillator system, in been isolated from microbiotas using the techniques of culturomics and
which the movement of each sub-pendulum influences the movement of metagenomics.
the other ones, resulting in the dynamics of the system becoming ne-
cessarily unpredictable and thus, chaotic. But just as a specific clone can
escape and spread in an environment, resistance genes can also become 2. Sources and factors influencing colonization
independent and diffuse. Finally, the selfish gene theory, developed by
Dawkins in 1976 [13], was proposed as an alternative to evolution 2.1. An ancient origin
theory. It puts the gene itself in the center of evolution. Thus, a gene
that confers an advantage for replication will survive in the next gen- ARGs have existed for a long time [16], long before the antibiotic
eration. It is not obviously translated in the phenotype and does not era that started in the 1930s with the use of sulfonamide. Some re-
inevitably provide an advantage for the organism. In Rickettsia species, sistance genes encoding resistance to β-lactams (bla gene), to tetra-
the presence of palindromic repeats in various genes [14] suggests that cycline (tetM gene) or to glycopeptides (vanX genes) were found in
this “selfish” sequence provides an advantage for protein sequence 30,000-year-old Beringian permafrost sediments [7]. Comparison of
creation. The creation of chimera genes between two pre-existing genes ancient and modern vanA genes and VanA protein structures confirmed
may also provide advantages for a gene that will spread across gen- the importance of the environment in the spread of antibiotic resistance
erations. The blaNDM gene, which encodes for a carbapenemase leading genes existing a long time before the antibiotic era [7]. Indeed, bacteria
to carbapenem resistance in bacteria, is an example of chimera between have always developed defense mechanisms to survive. They synthesize
a blaNDM pre-existing gene and an aphA6 gene [15]. The fusion of a part small natural molecules to defend themselves against other microbes as
of the sequence of aphA6 with blaNDM is thought to be responsible for well as antibiotic resistance mechanisms to protect themselves against
the spread of this resistance gene since 2008 [15]. Human microbiomes these same molecules [17,18]. Some serine β-lactamases have been
found in samples aged more than 2 billion years, even in plasmids,

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suggesting that horizontal gene transfer (HGT) existed long before an- demonstrated that resistance to fluoroquinolones in Escherichia coli was
tibiotic selective pressure and explaining why some bacterial pathogens linked to fluoroquinolone treatment [29,30]. These acquisitions seem to
are predisposed to acquire resistance [17]. The “antibiotic resistance come from an exogenous source. Moreover, it seems that resistance to
gene” function (for example, a metallo-betalactamase) would be a ex- fluoroquinolone is linked to methicillin-resistance, with a co-resistance
aptation of the ancestral function of the protein given by its structural of more than 80% in fluoroquinolone-resistant Staphylococcus.
specificity (a metallo-protease). The detection of a wide variety of re-
sistance genes in a pre-Columbian Andean mummy of the 11th century
[16] supports this hypothesis, as well as the exchanges between the 2.3. Natural environments, diet, animals and travel as sources of ARGs
human microbiome and its environment [7,19,20]. Similarly, an an-
cient sequence sharing only 30% similarity with a known protein ß- ARGs are very ancient and ubiquitous in nature, animals, diet and
lactamase found in a medieval sample was expressed in an Escherichia humans [7,31,32]. They can belong both to intrinsic or acquired re-
coli and showed a high level penicillinase activity [21], supporting the sistance phenotype for a particular bacterium as long as they are able to
hypothesis that ß-lactamases exists and are functional long before the increase the MIC of an antibiotic. They have been detected in regions
introduction of antibiotics. Even if in the natural environment mobile isolated from antibiotic exposure and with limited interaction with the
elements and resistance genes were already present, it seems that be- world and in the gut of isolated populations [24,31,33]. Despite the
fore the industrialized era, resistance genes in the human microbiome global presence of ARGs, diversity in bacterial microbiotas and the
were not clinically relevant and in low relative abundance, because of nature of ARGs are different according to the way of life and location of
the absence of a selective advantage for bacteria [17]. The arm race of people, and mainly diet dependent or environment dependent (Fig. 1).
bacteria against various release of heavy metals or antimicrobials is Thus, the fecal and oral microbiota of the Hadza of Tanzania and the
mainly driven by the environment and industrialization but those Amerindian Yanomami [33], who have hunter-gatherer lifestyles and
highly adapted bacteria in such environments may be selected in the live in isolated places, are more diversified than the microbiota of in-
human gut if there is a selective pressure from humans. Selection of dustrialized populations. This higher diversity can be explained by a
these bacteria in humans is not only due to specific weapons developed more significant environmental exposure and an alimentation that
by humans against those bacteria but could be also selected as a col- varies more frequently with seasonality than in industrialized countries,
lateral effect of other drugs [17]. Before this date, it seems that mobile suggesting that they are exposed to a higher diversity of bacteria thus a
elements existed, but they did not carry resistance genes. The spread of higher diversity of ARGs. Sequences of ARGs that have been detected in
ARGs is essentially mediated by integrons, especially class 1 integrons, isolated regions are very similar to those found in industrialized
which are carried by transposons and plasmids [22]. Class I integrons countries [31,33]. However, hunter-gatherer peoples have a robust and
are mobile elements that can capture cassettes of antibiotic resistance diverse type of ARGs, suggesting the environment (soil) as the origin of
using site-specific recombination mediated by integron-integrase (intI) some novel ARGs, while ARGs isolated in adults from industrialized
(Fig. 1). The source of integrons is not fully known, but they are derived countries mainly derive from diet. Studies have also shown that the
from environmental bacteria in sediments, and their expansion in relative quantity of ARGs is variable, depending on the country
human bacteria started with industrialization [22]. Their role in the [30–37]. Thus, it has been established that diversity of antibiotic re-
spread of antibiotic resistance is central, and they are present in sistance and the abundance of resistance genes are highest in Japanese
40–70% of Gram negative bacteria found in clinical practice, while they individuals, followed by southern Europeans (Spanish, French and
have just appeared in Gram positive bacteria [22]. After the appearance Italians), and Danish and American individuals. There is a greater
of the mer transposon responsible for resistance to mercury, other abundance of resistance genes in Chinese individuals compared to Ja-
compounds were used, forcing bacteria to constantly adapt and acquire panese individuals, but the diversity of ARGs was the lowest of all [35].
new resistance genes. This was the case for quaternary ammonium, This can be explained by different eating habits (fermented dietary
sulfonamides [23], and each time a new antibiotic has been marketed. products), antibiotic consumption or use of antibiotics in animal feed
The abundance of mobile elements is variable between individuals and [30–37]. Moreover, the gut microbiome of Chinese individuals carried
seems to be mediated by diet, which could favor horizontal gene some specific ARGs that were only detected in Chinese individuals, and
transfer (HGT) [24]. different ARGs conferring resistance for the same antibiotic [35].
Weight alterations might increase a number of bacterial populations,
2.2. Antibiotics modulate the abundance of antibiotic resistance genes more or less rich in ARGs, modulating the abundance of ARGs. It can
decrease after dieting in obese child [38] or increase in case of mal-
Different studies have shown various effects of antibiotic use on the nutrition, with an overabundance in pathogenic bacteria [35]. Anti-
gut microbiota, but in most cases, it reduces the relative abundance of biotic use in livestock also has an impact on the development of re-
microbes in the gut and increases the number of resistance genes. For sistance genes [2]. ARGs in human fecal samples are significantly
example, the use of macrolides in Finnish pre-school children causes a higher for antibiotics that were used in both animal breeding and
decrease in Actinobacteria and an increase in Bacteroides and human clinical practice or for antibiotics with an available analogue in
Proteobacteria [25]. Conversely, clindamycin administration reduces animal breeding. This significantly higher relative abundance of ARGs
the relative abundance of anaerobic bacteria such as Bacteroides and is also found for antibiotics available for a long time, and these two
increases the abundance of Enterobacteriaceae in infected adults [26]. In factors are found in different developed countries [34]. As lifestyle is
ARGs, the use of clindamycin or fluoroquinolones increases efflux pump different between communities, resistance gene acquisition in the gut
protein expression, which confers resistance to acriflavine, aminogly- microbiota can be mediated by international travel. Increase in re-
cosides, glycylcycline, macrolides and β-lactams. This protein was pri- sistance to cephalosporins by acquisition of a CTX-M and fluor-
marily detected in Proteobacteria, which showed a relatively abundant oquinolones by GyrA mutations has been detected in the gut resistome
increase under treatment [26]. However, beta-lactamase protein levels of healthy travelers after their return from the Hajj [39] or from India
were not modified after amoxicillin exposure [27]. Only efflux pump [40,41]. These destinations are known for their high rates of resistance
mechanisms leading to amoxicillin resistance were higher three months genes and their high risk of transmission of infectious diseases [39,41].
after amoxicillin exposure. It is well established that the use of fluor-
oquinolone in clinical practice selects fluoroquinolone-resistant strains.
Nasal acquisition of fluoroquinolone-resistant Staphylococcus occurs
both in community (42%) and hospitalized patients (94%), and is al-
most inevitable in hospitals [28,29]. In the gut, it has also been

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3. Evidence of asymptomatic carriage of bacteria carrying ARGs: A studies (Table 2) dealing with ARGs and the different microbiomes have
transmissible reservoir capable of becoming pathogenic allowed identification of genes that are predominantly represented in
the different microbiomes. These genes essentially involved antibiotics
3.1. Studying the human resistome used in clinical practice and are carried by species which are minor-
itarian in the intestine. Consequently, standard procedure fails to detect
As part of the Human Microbiome Project (HMP) project, numerous these ARGs, as they amplified genes that are mainly represented. Other
human samples from healthy or sick patients have been studied by methods such as targeted metagenomics have been developed to am-
metagenomic or culture analysis, highlighting the complexity of the plified thousand resistance genes with clinical impact, with the ad-
identification of ARGs [16,34,36,42,43]. These two methods are com- vantage to better detect these genes in the gut but with the incon-
plementary in detecting ARGs, but generally fail to identify new re- venient of a targeted procedure (as it amplifies only known genes) [60].
sistance mechanisms. Culture allows confirming the phenotype of an- The gut microbiota contains a wide variety of resistance genes, but
tibiotic resistance, but only a limited number of bacterial species can be those most frequently reported are especially directed against tetra-
studied on standard cultures [42]. It especially involves genes that are cycline, β-lactams, aminoglycosides and glycopeptides
carried by Proteobacteria and/or resistance genes that are directed [36,37,39,42,61,62], followed by chloramphenicol and macrolides
against antibiotics used in human health. Metagenomics contributes in [27,36,37,54,63]. Conversely, the respiratory microbiome contains
identifying resistance genes that belong to non-pathogenic or oppor- mostly β-lactamases [64,65]. Finally, the skin microbiota seems to be a
tunistic pathogens, and also unknown genes, for which their structures reservoir for β-lactamases, glycopeptides, aminoglycosides, fluor-
are likely related to a resistance gene, although their role in antibiotic oquinolones and macrolides-lincosamide-synergistins resistance genes
resistance is still unknown in phenotypic studies. The comparison of [66–68]. Among all these studies, efflux pump mechanisms represent a
sequences to antibiotic resistance genes databases such as Antibiotic ubiquitous resistance mechanism. Some resistance genes, such as those
Resistance Gene Online (ARGO) [44], MvirDB [45], Antibiotic Re- for tetracycline or chloramphenicol, are almost always detected in fecal
sistance Gene Database (ARDB) [46], Resfinder [47], the Comprehen- samples [27,35,36,69]. The reason for the high level of resistance to
sive Antibiotic Resistance Database (CARD) [48] or ARG-ANNOT [49] these two antibiotics is unclear. It may have been selected by intensive
allows to identify genes sharing homology with a current resistance agricultural use, or it may have a function in the gut microbiome that is
gene and to identify ancient resistance genes. The main problem with still unknown [27]. Thus, tetracycline resistance mechanisms in the gut
these methods is that they focus on ARGs directed against antibiotics microbiota are essentially major facilitator superfamily (MFS) efflux
used clinically [50]. For example, this was the case with the plasmid- transporters and ribosomal protection systems that are carried by Fir-
mediated mcr-1 gene, which encodes for phosphoethanolamine trans- micutes and Actinobacteria, which are mainly part of the commensal
ferase, leading to colistin resistance [51]. Colistin is an old antibiotic, flora [54]. The ribosomal protection enzyme is mainly represented by
used in the 1970s and then abandoned, which returned to use in the tetQ or tetW, depending on the country [36]. Concerning the erm-type
2000s because of its activity against multidrug resistant Gram negative conferring MLS resistance, ermB is the most frequent gene, followed by
bacteria [52]. In 2015, the mcr-1 plasmid-mediated colistin-resistant ermF and ermG. Concerning β-lactamases, mainly class A, class C and
gene was described for the first time [51]. This gene was soon found in class D β-lactamases have been detected in the gut microbiome, while
some strains dating from the 1980s [52] and in metagenomes [53], class B β-lactamases are more represented in soil microbiota [54,61].
showing that this gene had existed for a long time, but as the antibiotic Resistance to cephalosporins is primarily represented by Bl2e_cfxa,
was not used for a long period, its resistance was not evaluated. Indeed, Bl2e_cepa and Bl2e_cbla [36]. BacA (newly named UppP) is the most
it is difficult to find defense mechanisms directed against human arms frequent gene involved in bacitracin resistance, while vancomycin re-
that are unknown. sistance is mostly represented by the VanG operon [36]. The incon-
venient of standard metagenomic procedures is that it is difficult to rely
3.2. A dialogue between organisms ARGs and the species that are carrying them. The epic-PCR (Emulsion,
Paired Isolation and Concatenation PCR) [70] is a technique that has
Two different resistomes can be identified in human microbiotas, recently been developed to analyze the functional diversity of an en-
especially in the gut resistome: the resident resistome which is carried vironment as well as the functional role of each individual present in
by commensal flora, and the transitory resistome, carried by bacteria this particular ecosystem. The use of emulsion in this procedure allows
that are periodically present in the gut, but which can transfer their to separate a mass of cells into individual reactions by partitioned re-
resistance to commensal flora or become permanent bacteria of the action in drop whereas the concatenated PCR targeting both the ARG of
microbiota (Fig. 1). These modifications increase the gene pool of the interest and the bacterial rRNA 16S allows to identify the sequence of
resident resistome and promote exchanges with other bacteria. Some the gene and the species to which it is related. This procedure has been
resistance genes have been identified in commensal bacteria, such as mainly employed to study waste water but it could be applied on gut
CblA β-lactamases [54,55] in Bacteroides uniformis, tetQ in Bacteroides microbiome study [71,72].
taxa [56] or baca in Clostridiales taxa and the Streptococcus genus [26].
Various resistance genes found in the microbiome are highly similar to 3.4. Early acquisition of the human resistome
those providing potential resistance to chemotherapeutic agents in
human pathogens, suggesting that the microbiome constitutes a good The human resistome is acquired early in life. Some ARGs that are
reservoir for antibiotic resistance and for HGT from commensal bacteria present in healthy humans appear in infancy, soon after birth
to pathogenic bacteria [56,57]. As part of the gut microbiota, the re- [27,55,63,69]. Similar ARGs are found both in the infant and maternal
sident virome, through bacteriophages, can also contributes to the vagina or gut, suggesting maternal influence in the appearance of the
horizontal transmission of resistance genes [58] (Fig. 1). Phages can infant resistome [27,69]. However, the resistome of twins is sig-
thus be exchanged between commensal and pathogenic bacteria under nificantly more similar than with their mother or unrelated infants,
antibiotic selective pressure, but their range is less important because suggesting that the shared environment and host genetics may impact
they rarely are of broad host-spectrum. Transduction is generally ef- the gut resistome in the first months of life; more than the maternal gut,
fective if donor and recipients are closely related [59]. which, even if it has an impact on the gut microbiota, does not really
impact the resistome [27]. In these infants, resistance to β-lactams,
3.3. ARGs found in microbiota tetracycline, chloramphenicol, trimethoprim and cycloserine has been
detected, as well as resistance to colistin, tigecycline and aminoglyco-
The standard metagenomic studies (Table 1) and major culturomics side at a lower level [27,63,73]. ARGs are often transitory in the gut,

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Table 1
Summary of metagenomic studies concerning resistance in gut, oral, respiratory and skin microbiotas.
Reference Nature of microbiota Gene of resistance family identified

Gut & Oral Microbiota

Diaz-Torres et al., FEMS, 2006 [76] Oral microbiota Resistance to amoxicillin (8%), tetracycline (14.5%) and aminoglycosides (3.5%).
60 healthy adults Tetracycline: tet(M), tet(O), tet(Q). Less frequent: tet37 tet(W) tet(D and tet(A)
Sommer et al. Science. 2009 [42] Oral & Gut microbiota Tetracyclines (tetO)
2 unrelated healthy humans who did not β-lactams (CTX-M-15)
receive antibiotics for more than 1 year. 78 unknown resistance genes
Cheng et al., FEMS Microbiol Lett, Gut microbiota β-lactams: class A β-lactamase ORN-1 (Raoultella ornithinolytica), HGA-1, cbla-1 + unknown
2012 [61] 4 healthy unrelated volunteers class D β-lactamase.
Kanamycin: AAC(6′)- APH(2″) (Staphylococcus pseudintermedius), unknown AAC(6′)
D-cycloserine: D-ala-D-ala ligase

Tetracycline: tetO, tetW, tet40, tet32

Forslund et al., Genome research, Gut microbiota Resistance to 50 of 68 antibiotics screened.
2013 [34] 252 American, Danish & Spanish Average of 21 ARGs per sample.
Antibiotic resistance potential variable according to country.
Shankar Ghosh et al., PloS One, Gut microbiota Tetracycline resistance (267/275): Bacteroidetes and Clostridiaceae
2013 [35] 275 American, Danish, French, Bacitracin resistance (263/275): bcra, babca. (Firmicutes, Bacteroidetes, Proteobacteria)
Italian, Spanish, Japanese and Chinese Vancomycin resistance (255/275): vanug, vang, vanrg. (Firmicutes)
individuals + Cephalosporins resistance
Gut metagenomes of healthy and MLS resistance: F3H8F5 profile (182/275)
malnourished and β-lactams, fosmidomycin, chloramphenicol, sulfonamides, trimethoprim resistance
children from India
Hu et al., Nature communications, Gut microbiota 1093 unique antibiotic resistance genes
2013 [36] 162 healthy individuals In all gut microbiota (162): Ant6ia, BacA, VanRA, VanRG, Tet32, Tet40, TetO, TetQ and
TetW; ErmB (161/162)
Tetracycline resistance TcR: Tet37 in 94% individuals, Tet36 (first identified in Bacteroides
from swine manure pits) in 16% of individuals.
Moore et al., PloS One, 2013 [63] Gut microbiota β-lactamase: classes A (TEM, SHV, CTX-M, and VEB protein families), C, and D in all ages and
22 fecal samples from healthy patients from B in infants.
1 month to 19 years old. Aminoglycoside: AAT, APT and AAD
Chloramphenicol: acetyltransferases, MFS- chloramphenicol transport proteinTetracycline
and Tigecycline: Ribosomal protection proteins, MFS and MATE transport proteins, and flavin-
dependent monooxygenases. Multiple tet genes(tetQ++), flavin dependent monooxygenases
(TetX1 and TetX2);
D-cycloserine: D-ala-D-ala ligaseSulfonamides: dfrA
Regulator of efflux pump protein: marCRAB locus, soxR, soxS
Fouhy et al., Plos one, 2014 [73] Gut microbiota β-lactams: blaTEM, blaOXA, blaROB and blaCTX-M genes (E. coli, Shigella and Serratia gene
22 healthy infants who had not been treated homology)
with antibiotics Aminoglycosides: ant (20)-Ia, aph (20)-Id, aac (69)-Ie-aph (20)-Ia (E. coli, E. faecalis, S.
epidermidis and C. difficile)
Von Wintersdorff et al., EID, 2014 Gut microbiota CTX-M acquisition: 9 before to 33,6% after travel (p < 0.001)
[41] 122 healthy travelers’ fecal samples Quinolone: qnrA (0.8 to 3.3%), qnrB, (6.6 to 36.9%) and qnrS (8.2 to 55.7%) (p < 0.001)
High prevalence before and after cfxA, tetQ, tetM, ermB and aac(6′)-aph(2′)
Warinner et al., Nat Genet, 2014 Ancient Dental microbiota Multidrug efflux pumps and native resistance genes to aminoglycosides, β-lactams,
[31] 4 adults bacitracin, bacteriocins, and macrolides.
Gibson et al., The ISME Journal, Gut microbiota 6179 microbial proteomes of β-lactams, aminoglycosides, fluoroquinolones, glycopeptides, macrolides, tetracyclines
2015 [54] soil and human microbiota as well as efflux pumps and transcription factors modulating AR.
Moore et al., Microbiome 2015 Gut microbiota β-lactams, tetracycline, chloramphenicol, trimethoprim and cycloserine
[27] 3 healthy twin pairs
18 infants + 3 maternal
Santiago-Rodriguez, Plos One, Gut microbiota Sequences associated with putative beta-lactamases, penicillin-binding proteins, resistance
2015 [16] Paleofeces, and colon samples of an 11th to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfonamides,
century CE pre-Columbian Andean mummy quinolones, tetracycline, vancomycin and multidrug transporters
Glycopeptides: sequences sharing homology to vanA, vanB, vanH, vanT, vanY, vanR and vanS
Clemente et al., Sci. Adv. 2015 [33] Fecal and oral microbiota Pool of Amerindian E. coli β-lactams: ampC
4 Yanomami health workers from an isolated, isolates from fecal samples. Efflux pumps: mdfA, bcr, mdlB, mdlA, marA, rob, soxS,
previously uncharted village in the High Amerindian microbiota Fecal AraC family transcriptional regulator
Orinoco state of Venezuela. β-lactams: cbla gene
Chloramphenicol: cat
Tetracycline: tetW
MATE transporter
Oral Penicillin binding-protein
Chloramphenicol: cat
Efflux pumps: MFS and ABC transporters,
emrB
Puerto Rican microbiota Fecal β-lactamase
tetracycline: Ribosomal protection
protein, TetX
Oral β-lactamase class A, class B3, class B1,
penicillin binding protein.
Efflux pump: MFS transporter, emrB, bcr/
cflA
(continued on next page)

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Table 1 (continued)

Reference Nature of microbiota Gene of resistance family identified

Rampelli et al., Curr Biol, 2015 Gut microbiota Tetracycline resistance (MFS):
[24] Hadza hunter-gatherers Tet(M)-Tet(W)-Tet(O)-Tet(S)
β-lactamases
Lincosamide, glycopeptide, polypeptide,
multidrug resistance
Gut microbiota Bacitracin, Tetracycline, Erythromycin,
Western urban Italian adult Aminoglycosides, Chloramphenicol, Polypeptide
Yang et al., Gene, 2016 [37] Gut microbiota Tetracycline: Tet-36, Tet-37, TetB-P, Tet-40, Tet-44, otrA, Txcr3, TetM, TetO, TetQ, TetS,
1267 human stool samples from 4 countries in TetT, TetW, Tet-32, Tet-35.
3 continents Glycopeptides: VanA-G,VanS-Pt2,VanA-L, VanS-A,VanS-B,VanS-C, VanH-Pt, VanS-D, VanRc3,
Vans-F, VanRc4, VanS-L, VanS-M,VanY-D,VanH-Ac1,VanR-Pt,VanH-A,VanH-B,VanH-D,VanH-
M, VanH-Pt2,VanS-Pt,VanSc3,VanSc4,VanW-B,VanZ-A,VanE,VanH-Ao2,VanT,VanR-B, VanR-
C, VanR-Pt2,VanR-D,VanZ-Pa,VanR-F,VanTc,VanTc4,VanR-L,VanR-M,VanX-D,VanA-D
MLS: OleB, OleC, SrmB, ErmC, LnuA, CarA, LnuC, Erm31, Erm32, LmrB, Erm39, VatB, VatE,
VgaA, VgaB, VgaD, VgaE, MefA, LsaA, LsaB, LsaC, MsrC, MsrD,TlrC
β-lactamases Metallo_beta_lactamase,PENA,HugA,Zn-dependent_hydrolase,OXA-53,TLA-
1,OXA-256, PBP_EC,MECA,AmpC,AmpC1_EC,CEPA-44,SFO-1,Beta-lactamase_class-A
Fluoroquinolones: QnrC,OqxBgb,norA,QnrB48,OqxA
Sulfonamides: SulIII,SulII
Phenicols: CmrA,CmlV,CatA3,CatB1,CatB2,Cfr
Aminoglycosides: Spc,Aph3-III,Ant6-Ib3
Trimethoprim: DfrA3,Dfr22
Gosalbes et al, J Dev Orig Health Gut microbiota Meconium: ARGs detected in 14 (70%) of meconium samples. 10 (50%) of the samples carried
Dis, 2016 [55] 20 infants followed from birth to 1 year old BLr, 8 (40%) carried Tcr and 4 (20%) carried both.
β-lactamases: mecA and blaCTX-M in 9 (64%) and 3 (21%) samples.
Tetracycline: 8/11 tested ARGs. Each of these genes was only detected in one [tet(C), tet(K),
tet(Q)] or two samples [tet(A), tet(B), tet(L), tet(O), tet(X)].
Pehrsson et al., Nature, 2016 [43] Gut microbiota 121 novel proteins ( < 60% aa identity to any protein in NCBI nr), of which (72%) predicted
51 humans as antibiotic modifiers, including 57 class A β-lactamases.

Skin microbiota
Mathieu et al., Plos one, 2013 [77] Skin microbiota Methicillin resistance in Staphylococci
2 healthy males Bacitracin and teicoplanin resistance sequences
Oh et al., Nature, 2014 [66] Skin microbiota Significant variability of quantity and resistance type between both individual- and site-
15 healthy people samples.
Broadly represented: class A beta-lactamases, rRNA methyltransferases, efflux
mechanisms, or quinolone resistance.
Host specific: multi-antimicrobial extrusion (MATE) efflux pumps.
Site-specific dominance: lincosamide resistance in foot sites.
Oh et al., Cell, 2016 [67] Skin microbiota OTUs related to β-lactam resistance, vancomycin resistance.
27 healthy people

AA: amino acid; ARGs: Antibiotic Resistance Genes; MLS: macrolide-lincosamide-streptogramin; AAT: Aminoglycosides acetyltransferase; APT: Aminoglycosides
phosphotransferase; AAD: Aminoglycosides adenylyltransferase; ESBL: Extended Spectrum Beta-lactamase; MFS: Major facilitator Superfamily; MATE: multi anti-
microbial extrusion; GN: Gram negative; GP: Gram positive.

and some mechanisms of resistance, especially the efflux pump, tend to pathogens such as Achromobacter xylosoxidans, Stenotrophomonas mal-
decrease with increasing age [27]. tophilia or non-tuberculous Mycobacterium are also more frequently
found in these patients. The persistence of bronchopulmonary infec-
tions exposes the patient to frequent and varied use of antibiotics, as
4. The resistome and pathology: Example of cystic fibrosis well as to the acquisition of resistant bacteria. The frequency of colo-
nization by multidrug resistant Pseudomonas aeruginosa has been esti-
Cystic fibrosis is an autosomal recessive disease that affects re- mated at 45% [64]. The use of antibiotics may also select some resistant
spiratory, hepatobiliary, pancreatic and lower gastro-intestinal tracts anaerobic strains of the microbiota, such as Prevotella species, which
[74]. Recent studies have shown a common core microbiota between were found to be more frequently resistant to amoxicillin + clavulanic
gut and respiratory tract microbiomes in early age, highlighting the acid than strains belonging to healthy patients [65]. Various mechan-
possibility of an interaction between these two microbiotas [75]. isms of resistance have been found in these bacteria. Efflux pumps are
Moreover, some modification in the gut and respiratory microbiota frequently observed, especially in P. aeruginosa, and lead to resistance
precede Pseudomonas aeruginosa’s first respiratory colonization, with a to several antibiotics, such as tetracycline or β-lactams. The erm-type
significant decrease in Parabacteroides in the gut and an increase in the genes have been found in P. aeruginosa, H. influenzae, Mycobacteria,
relative abundance of Salmonella in the respiratory tract observed [75]. Staphylococcus or Streptococcus. β-lactamases genes are also found in the
P. aeruginosa, was in this study significantly associated with the lung sputum of cystic fibrosis patients: AmpC has been found in P. aerugi-
microbiota and a previous intestinal colonization was not found. nosa, the expression of mecA in S. aureus, and other types of β-lacta-
However, the study from Madan et al. suggests an association with mases are carried by Burkholderia cenocepacia or anaerobic bacteria of
nutritional changes prior to first acquisition of P. aeruginosa, whose the microbiota, such as Prevotella sp or Bacteroides [64,65].
origin in respiratory tract remains unknown. Modification of the re-
spiratory tract promotes recurrent bacterial infections and the frequent
use of antibiotics, leading to the emergence of multidrug resistant 5. Conclusion
bacteria. Staphylococcus aureus, Haemophilus influenzae, Pseudomonas
aeruginosa or Burkholderia cepacia complex are pathogens frequently Human microbiomes are a wide source of ARGs and a potential
found in the sputum of cystic fibrosis patients [64]. Some opportunistic reservoir for pathogenic bacteria becoming more resistant. Various

48
 Table 2
Summary of culture studies concerning resistance in gut, oral, respiratory and skin microbiotas.
Reference Nature of microbiota Multidrug resistant bacteria found Gene of resistance family identified
S.A. Baron et al.

Gut & Oral Microbiota

Gueimonde et al., FEMS Immunol Med Gut microbiota Bifidobacterium high and TetW in mother tetM (both GN and GP bacteria) the more frequent in both, followed by tetO and
Microbiol, 2006 [69] 29 healthy breast-fed infants + 20 Clostridia low and TetW in infants. Idem for TetO tetW (Fusobacterium prausnitzii and Bifidobacterium longum)
mothers of the infants
Pallecchi et al., Antimicrob Agents Gut microbiota Enterobacteria Fluoroquinolone: qnrB (54%) mostly in E. coli, qnrS (15%) mostly in Klebsiella
Chemother, 2009 [78] 310 healthy children from Bolivia pneumoniae
and Peru
Alicea-Serrano et al., Arch. Microbiol, Oral and fecal microbiota PCR Infants with vaginal tetM and tetO only in mouth
2013 [62] 10 newborns delivery
Vagina and mouth of their mothers Infants with Cesarian tetO in mouth and tetW in mouth and meconium.
section-delivery
Mothers tetM, tetO, sometimes tetQ (vagina) and tetW
Culture 572 bacteria Tetracycline efflux pumps (TetA)
Aminoglycoside: AAC(3)-II, AAC(6)-Ib, β-
lactams: TEM, AmpC, and CTX-M,
CblA, CfxA
Woerther et al., Clin Microbiol Rev, 2013 Gut microbiota Before 2008: < 10% ESBL Enterobacteria fecal carriage in all regions of the world
[79] Europe: max 11.6% fecal carriage in a geriatric unit in Belgium
Eastern Mediterranean: 7.3% in Tunisia in 2010, 63.3% in Egypt in 2011
Africa: 10% in Senegal to 30.9% in Niger
Southern Asia: max 69.3% in 2010 in Thailand
Western Pacific: 50% in Fujian in 2009
Americas: Latin Americas 12.4% in 2011
Predominance of CTX-M ESBL genes (CTX-M-15, CTX-M-14 in Southeast Asia, CTX-M-2 in South America)
Johnning et al., BMC Microbiology, 2015 Gut microbiota Escherichia coli Increase in the quantity of S83L amino acid substitution in GyrA, from 44% to

49
[40] 12 student travelers in India + 12 72% (p = 0.036).
Indian resident fecal samples
Narimani et al., Microb Pathog, 2016 Gut microbiota 177 Bacteroides β-lactams: 95.4% resistance to ampicillin
[80] 230 healthy individuals Clindamycin: 95%, Erythromycin 93% ermF
Tetracycline: 86% tetQ gene
Ciprofloxacin: 74%
Tellevik et al., PloS One, 2016 [81] Gut microbiota Enterobacteria ESBL carriage:
250 healthy community children - Healthy: 29 (11.6%)
250 with diarrhoea 103 other - Diarrhea: 118 (47.2%)
diseases - Other diseases: 60 (58.3%)
139 K. pneumoniae, 129 E. coli, 11 E. cloacae
Farra et al., Clin Microbiol Infect, 2016 Gut microbiota Enterobacteriaceae 59% (79/134) positive sample whom 83/134 ESBL-Enterobacteriaceae
[82] 134 healthy children from Bangui ARGs: 81/83 blaCTX-M-15 frequently associated with qnr (54/81, 66%) and aac
(Central African Republic) (60)-Ib-cr (35/81, 43%) genes.
Leangapichart et al., AAC, 2016 [39] Gut microbiota Escherichia coli: 14 to 28% for any antibiotics (p = 0.0009), 3.9 to 14% CTX-M acquisition: 10.08% (13/129) before to 32.56% (42/129) of return
129 Pilgrims during the Hajj, (p = 0.008) for ceftriaxone and ticarcillin-clavulanic acid (12.4% versus samples (P < 0.001)
rectal swabs 22.5%; P = 0.048)
K. pneumoniae: 17,1 to 18,6% for any antibiotics
Ny et al., J Antimicrob Chemother, 2017 Gut microbiota E. coli Carriage rate of 4.7% (93 ESBL and 8 plasmidic AmpC)
[83] 2134 fecal samples from Swedish Genes: CTX-M-15 (45.3%), CTX_M-14 (20%), CTX-M-1 (11.6%) and CTX-M-27
inhabitants (8.4%)

Respiratory microbiota
Sherrard et al, The Lancet, 2014 [64] Respiratory microbiota Pseudomonas aeruginosa Efflux pumps (P. aeruginosa), erm-type genes (P. aeruginosa, H. influenzae,
Sputum of cystic fibrosis patients Antibiotic resistant small colony variant Staphylococcus aureus Mycobacteria, Staphylococcus, Streptococcus)
A. xylosoxidans β-lactamases AmpC (P. aeruginosa)
Burkholderia cepacia complex Other β-lactamases (Burkholderia cenocepacia, Prevotella sp, Bacteroides)
Prevotella sp MecA (S. aureus)
(continued on next page)
Human Microbiome Journal 10 (2018) 43–52
S.A. Baron et al. Human Microbiome Journal 10 (2018) 43–52

MLSB resistance phenotype observed in 73/99 − > presence of the erm (X) gene

AA: amino acid; ARGs: Antibiotic Resistance Genes; MLS: macrolide-lincosamide-streptogramin; AAT: Aminoglycosides acetyltransferase; APT: Aminoglycosides phosphotransferase; AAD: Aminoglycosides adenylyl-
factors can modulate the human resistome, including the environment,
diet, antibiotics, lifestyle and travel. Chronic infections, such as cystic
fibrosis, with all the associated factors previously cited, are also the
38/50 (76%) ESBL positive including 23 (61%) resistant to clavulanic

cause of acquisition of resistance. Metagenomic studies have increased

the identification of new, unknown genes that do not belong to common
pathogenic bacteria but to commensal bacteria [42,61]. Our knowledge
about antibiotic resistance is still poor and only partially understood at
present. This lack of knowledge also applies to factors involved in
transfer between bacteria, especially in the incompletely known com-
plex ecosystem, and limits our understanding of the exchange and
communication that exists between bacteria [42]. Further studies are
warranted to identify both the putative ARGs present in the human
Gene of resistance family identified

microbiome, as well as the dynamics of their acquisition/exchange in

22/50 (44%) positive for blaTEM

the human microbiome. Moreover, we also need to identify them to

discover molecules that are synthesized in the human microbiome that
transferase; ESBL: Extended Spectrum Beta-lactamase; MFS: Major facilitator Superfamily; MATE: multi antimicrobial extrusion; GN: Gram negative; GP: Gram positive

may contribute to the selection of microbes that are associated either

with symbiosis and health or dysbiosis and disease.
acid + amoxicillin.

in all strains.

6. Financial support

This work was supported by the French Government under the «

Investissements d’avenir » (Investments for the Future) program man-
aged by the Agence Nationale de la Recherche (ANR, fr: National
Agency for Research), (reference: Méditerranée Infection 10-IAHU-03).
This work was supported by Région Provence Alpes Côte d’Azur and
European funding FEDER PRIMI.

Conflict of interest

None to declare.

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