Eﬀects of Antibiotics upon the Gut Microbiome:
A Review of the Literature
Theocharis Konstantinidis 1, Christina Tsigalou 1, Alexandros Karvelas 1,
Elisavet Stavropoulou 2, Chrissoula Voidarou 3and Eugenia Bezirtzoglou 4, *
1Laboratory of Microbiology, Medical School, Democritus University of Thrace, Dragana,
68100 Alexandroupolis, Greece; firstname.lastname@example.org (T.K.); email@example.com or
firstname.lastname@example.org (C.T.); email@example.com (A.K.)
Centre Hospitalier Universitaire Vaudois (CHUV), Rue du Bugnon, Vaud, CH-1011 Lausanne, Switzerland;
3Public Health Laboratory, Arta Prefecture, 47100 Arta, Greece; firstname.lastname@example.org
4Laboratory of Hygiene and Environmental Protection, Medical School, Democritus University of Thrace,
68100 Alexandroupolis, Greece
*Correspondence: email@example.com or firstname.lastname@example.org
Received: 19 September 2020; Accepted: 13 November 2020; Published: 16 November 2020
The human gastrointestinal tract carries a large number of microorganisms associated with
complex metabolic processes and interactions. Although antibiotic treatment is crucial for combating
infections, its negative eﬀects on the intestinal microbiota and host immunity have been shown to be
of the utmost importance. Multiple studies have recognized the adverse consequences of antibiotic
use upon the gut microbiome in adults and neonates, causing dysbiosis of the microbiota. Repeated
antibiotic treatments in clinical care or low-dosage intake from food could be contributing factors in
this issue. Researchers in both human and animal studies have strived to explain this multifaceted
relationship. The present review intends to elucidate the axis of the gastrointestinal microbiota and
antibiotics resistance and to highlight the main aspects of the issue.
Keywords: antibiotics; resistance; gut microbiome; microbiota
Undoubtedly, since the discovery of penicillin by Alexander Fleming in 1928 and thereafter,
antibiotics used in the management of infectious diseases have saved millions of lives [
]. Over the
past two decades, antibiotic misuse and overuse has come to be considered a serious public health
issue, imperiling the great achievements of medicine. Antimicrobial resistance (AR) is a developing
concern that threatens to harm the eﬀective treatment of infectious diseases, especially in high-income
]. Even though the irrational use of antibiotics was once considered a problem only in
developed countries, a striking rise in low and middle-income countries has occurred [
]. It is of
interest to note that de Jong et al., in their observational study, revealed that antibiotic-resistant
bacteria from animal farms could be the reason for therapeutic failure in adults living in rural areas,
an assumption that of course needs further investigation [
]. However, another study described that
higher prevalence rates of 8-fold were observed in urban settings when compared to rural settings, as
antibiotic prescription was more frequent in towns with hospitals. The antibiotic prescription rate
in urban areas was 46.8% where people receive more qualiﬁed hospital care [
]. AR occurs more
frequently in hospitals due to the increasing number of patients, surgical procedures, and interventions,
which are linked to the increasing use of antibiotics in the health care setting.
Antibiotics resistance percentages are rising daily, not only concerning the hospital community,
but also various other territories. Antibiotics are given to animals for treating infections, but mostly to
Biomedicines 2020,8, 502; doi:10.3390/biomedicines8110502 www.mdpi.com/journal/biomedicines
Biomedicines 2020,8, 502 2 of 15
achieve faster growth for commercial purposes. Moreover, AR is also present in plant pathogens [
Antibiotics used for therapy and animal feeding contribute to the spreading of antibiotics resistance in
food and environment [
]. Moreover, ESKAPE (Enterococcus,S. aureus,K. pneumoniae,A. baumannii,
P. aeruginosa, and E. coli) pathogens are a major cause of hospital-acquired resistant infections worldwide,
as they are associated usually with more serious morbidity and mortality rates and consequently to an
important economic loss due to various complications, prolonged hospitalization, expensive drugs,
and absenteeism in workplaces .
It is well known that antibiotics can produce alterations in the host’s indigenous microbiota by
selecting resistant bacteria that can appear as opportunistic pathogens [
]. Additionally, a low-dosage
intake of antibiotics or sub-therapeutic antibiotic treatment (STAT) from food and the environment have
also been associated with gut dysbiosis. Gut dysbiosis promotes negative eﬀects in plenty of systems
and functions of the host. Since the gut microbiome could be “at the intersection of everything”,
its alterations have been linked to multiple pathological conditions, and scientists have focused on
the relationship between antibiotics and the gut microbiota [
]. Accumulating evidence mainly from
animal studies has underscored the contribution of antibiotics to gut microbiome disruptions [
Although morbidity and mortality, due to infectious diseases, were remarkably reduced, antibiotic
treatment has been implicated in gut microbiota disruptions.
Nowadays, resistance represents a common trait for almost all developed antibiotics.
Unfortunately, at the end of 20th century the development of new antibiotics was dramatically
decreased due to economic and regulatory obstacles .
In this review, we summarize current evidence regarding the gut microbiome and its alterations
in relation to antibiotics, analyzing the reasons associated with their inappropriate use.
2. Insights to the Gut Microbiome
Until now, the usage of classic microbiological techniques has limited the amount of information
found about the human microbiome. However, the introduction of new molecular methods such
as next-generation sequencing (NGS) and methodologies such as 16S ribosomal RNA (rRNA) gene
sequencing and metagenomic shotgun sequencing have revolutionized scientists’ knowledge about
The abundance, diversity, and features of microorganisms’ genes are collectively known as
the human microbiome, a seemingly “new actor on stage” due to its numerous roles in health
and disease [
]. Several publications have demonstrated the relationship between dysbiosis and
inﬂammatory and metabolic diseases, such as inﬂammatory bowel disease (IBD), obesity, cancer,
asthma, autism, autoimmune diseases, etc. [
]. Until now, these studies have failed to establish a
causative role for the microbiome but have mainly focused upon the relationship between pathogenesis,
clinical manifestations, and disease prognosis with microbiome alterations.
The human microbiome is comprised of almost 40 trillion bacterial cells and about 30 trillion
human ones, revising the notion of the ratio closer to 1:1 [
]. Most microbes belong to ﬁve major
phyla: Firmicutes,Bacteroidetes,Actinobacteria,Proteobacteria, and Verrucomicrobia [
]. The gut holds
the majority of species—around 2000, with Bacteroidetes and Firmicutes representing more than 90%
of its microbes [
]. The gut microbiome contributes to human body functions such as digestion,
metabolism, protection from pathogenic microbes, the production of vitamins, as well as the regulation
of the immune system and inﬂammatory reactions. These functions represent those of an “active
organ”  or a microbial “endocrine organ” [18,19].
The human gut microbiome has been categorized into three enterotypes according to the variation
in gut microbes [
]. A person’s enterotype could change due to diﬀerent factors such as gender,
age, food intake, vaccinations, infections, smoking, etc., resulting in diﬀerences in the composition
and diversity of the gut microbiota from newborns to elders [
]. The gut is massively colonized
after birth, excluding the possibility that the fetal gut is sterile [
]. Moreover, it was shown that
the composition of the human microbiome is aﬀected by age and comorbidities [
]. A “healthy” gut
Biomedicines 2020,8, 502 3 of 15
microbiome has a high diversity; any kind of disruption may lead to dysbiosis, a critical condition
of imbalance between commensal and pathogenic microbes [
]. Eubiosis due to beneﬁcial bacteria
maintains an important homeostatic niche by preventing any disequilibrium that might cause dysbiosis
and, consequently, metabolic and inﬂammatory conditions, including asthma, obesity, cancer, autism,
and autoimmune diseases .
As mentioned, a speciﬁc diet may shape the proﬁles of gastrointestinal bacteria in humans.
Diﬀerences in food intake create a diﬀerent community structure of the gut microbiota [
]. For example,
comparing the microbiota of European children (EU) to children coming from a rural African village
in Burkina Faso (BF), an environment that resembles that of Neolithic farmers, by high-throughput
16S r DNA sequencing and biochemical analysis [
] one could see that BF children presented a
higher proportion of Bacteroidetes numbers than Firmicutes. Moreover, Prevotella spp. and Xylanibacter
spp. were prevalent in these children; both are involved in cellulose and xylan hydrolysis. However,
these bacteria were absent from the intestinal microbiota of the EU children. Additionally, the BF
children showed a higher ability to produce metabolites such as short-chain fatty acids (SCFAs).
The microbiota of BF and EU children has co-evolved with diet since ancient times, and the high
amounts of SCFA seemed to provide the host with an important amount of energy [
]. In both
populations, Actinobacteria with a predominance of the genus Biﬁdobacterium were present in younger
infants who were breast-feeding [7,26].
The characteristic proﬁle of the newborn gastrointestinal microbiota depends on age, race,
and the subject’s diet [
]. Several hours after birth, the newborn develops its normal microbiota.
Colonization by Biﬁdobacterium happens within four days after birth. Breast-fed infants carry a typical
gut ﬂora featuring an increased concentration of Biﬁdobacterium. However, infants receiving artiﬁcial
alimentation do not usually carry Biﬁdobacterium or demonstrate low concentration numbers, showing
a generally lower microbial diversity. Moreover, male newborns show a higher count of Biﬁdobacterium
than females. Nevertheless, in both sexes its preponderance is manifested after maternal alimentation.
Positive eﬀects of Biﬁdobacterium sp. on infant growth and health status have been reported [
]. A ﬁerce
competition has been exhibited between B. biﬁdum and C. perfringens in the gut of newborns delivered
by caesarean section [
]. Multiple authors have stated the beneﬁcial action of several bacteria on the
intestinal ecosystem, amongst them Biﬁdobacterium spp. [35–38].
By the time the child reaches the age of three or four years, two dominant phyla exist:
Firmicutes and Bacteroidetes. The Firmicutes phylum includes Lactobacillus,Bacillus,Clostridium,
Enterococcus, and Ruminicoccus genera, which may exhibit diametrically opposite actions. For example,
Faecalibacterium prausnitzii is more abundant in the gut of obese children than in non-obese
children, whereas Clostridia in human feces are associated with s lower body mass index. Moreover
E. feacalis escaping from the gut might cause a deleterious blood infection, while Bacteroidetes are
]. However, the diﬀerences were related to the presence of the Firmicutes
phylum’s class, the Mollicutes class, as obtained by animal studies with diet-induced obesity [
From this point forward, the gut microbiota tends to maintain a well-balanced condition, with few
changes across the adult life, ending in a diﬀerent state in the elderly [
], who show a decrease in
Biﬁdobacterium spp. [
]. Diet and drugs correspond to critical microbiome alterations, when other
factors, such as genetics, have less impact on the microbial population .
In the human intestine, bacterial levels rise along the intestinal lumen. For example, the bacterial
numbers can be as high as 10 million bacteria/mL in fecal ﬂuid. Qualitative and quantitative
diﬀerentiation is registered in bacterial populations colonizing diﬀerent parts of the gastrointestinal
]. Lactobacillus, which are facultative anaerobic or aerobic rods, are permanent residents
of the ecosystem of the human gut [
]. Diﬀerent studies suggest that the advantageous eﬀects of
Lactobacillus are strain-dependent. Agerholm-Larsen L et al. reported weight gain with the use of L.
rhamnosus and also with L. acidophilus [
]. On the other hand, L. gasseri BRN17 and L. gasseri SBT2055
in diﬀerent studies are associated with weight loss [
]. Lactobacillus show a selective adherence to
the intestinal epithelial cells [
]. Enterobacteriaceae are associated with gastrointestinal infections
Biomedicines 2020,8, 502 4 of 15
and carry speciﬁc adhesins, which mediate their adhesion to the intestinal mucosa [
non-pathogenic anaerobic bacteria, such as Lactobacillus and Biﬁdobacterium, could impede the ability
of the adhesion and invasion of several enteropathogenic enterobacterial strains .
In addition to food-induced eﬀects on the gut microbiome, a signiﬁcant contribution to its
development is derived from the administration of probiotics, prebiotics, and antibiotics [
and prebiotics might oﬀer a more balanced protection in the gut, but antibiotics might decrease diversity
and promote dysbiosis [
]. Another factor that might decrease diversity and promote dysbiosis is
alcohol abuse [
]. Nevertheless, the explicit factors deﬁning the development of beneﬁcial lactic acid
microbiota are not perfectly clariﬁed, but research focusing on the distributions of diﬀerent strains
in the various human organs, during states of health and disease, may elucidate them. Adequate
knowledge of the intestinal microbiota and its probiotic proﬁle in health and disease could provide
therapeutical advancements. Therefore, the probiotic approach will assist the investigation of the role
of bacterial species, as well as those components promoting their growth in the human intestine.
3. Antibiotic-Associated Shifts in the Gut Microbiota
As soon as antibiotics were introduced, they were acknowledged as the most eﬀective and
life-saving drugs to combat infectious diseases, and they resulted in a substantial decrease in morbidity
and mortality. However, humanity soon realized that the irresponsible and thoughtless use, misuse,
and overuse of antibiotics led to the emergence of antimicrobial resistance (AR). AR poses a global threat
to modern medicine and its achievements and is a major health problem [
]. Additionally, recent
studies have illuminated the potential impact of antibiotic intake on the intestinal microbiome.
Antibiotics can negatively aﬀect the required diversity of the gut microbiota in adults [
and children [
]. The short-term eﬀects of antibiotic use include diarrhea, Clostridium diﬃcile
infection, and AR [
], whereas the long-term consequences include the development of allergic
conditions—namely, asthma or food allergies and obesity [65,66].
Antibiotic administration for therapeutical purposes aﬀects the bacterial microbiota both
quantitatively and qualitatively by reducing or eliminating bacterial species and allowing other
species to obtain more space and nutrients in the intestine. This microbial imbalance inﬂuences the
state of health and disease. However, these studies have faced limitations, such as drug composition
and route of administration, as well as the age of the patient, the deleterious impact of antibiotics
in early life [
], and other factors such as diet and functional foods [
]. In particular, Cox et al.
introduced the concept of the “critical developmental window” in the early life of mice when low-dose
antibiotics had the greatest impact on the gut microbiome, leading to metabolic eﬀects .
The impact of antimicrobial agents used therapeutically or as a prophylaxis on normal
gastrointestinal microbiota causes disturbances in the ecosystem’s equilibrium. In all cases,
disequilibrium and alterations in the microbiota ecology depend on the involved drug and its
pharmacokinetic proﬁle [
]. The human intestine has the capacity to metabolize drugs due to the
possession of an enormous carriage of Cytochrome P450 (CYP) enzymes, which are responsible for
the catalyzation reactions in phase I and phase II of drug metabolism [
]. Korpela et al. have
demonstrated that oral antibiotic therapy with macrolides led to changes in the intestinal microbiota
by creating a shift in the relative abundance of Bacteroides and Biﬁdobacterium [
]. In the same vein,
antibiotic treatment breaks the intestinal equilibrium, leading to a niche perfect for C. diﬃcile growth
and spore germination [
]. However, other authors have stated an antibiotic-induced rise in toxin
production by C. diﬃcile as a stress-induced response that may vary following the bacterial strain [
Likewise, antibiotic abuse lead to negative eﬀect on the levels of proliferation or apoptosis of
intestinal cells, to enterocytes (sucrase) and endocrine cells. Therefore, lots of intracellular proteins
are released. Except for the local functional activity of releasing proteins, they may be useful as
markers of gut microbiome dysregulation. Zhernakova et al., in their study of the gut microbiota
of 1135 participants from a Dutch population-based cohort, demonstrated a connection between the
Biomedicines 2020,8, 502 5 of 15
microbiome and the diﬀerent host factors. Authors reported that fecal chromogranin A (CgA) was
exclusively associated with the presence of particular microbial species .
Immunomodulatory and Indirect Eﬀect of Antibiotics on the Gut Microbiota
The eﬀect of antibiotic drugs to the human microbiome is complex and bi-directional. Except
for direct eﬀect, antibiotics can also indirectly aﬀect human microbiota. The gut microbiota dysbiosis
following exposure to antimicrobial agents may cause the dysregulation of immune responses [
Indeed, it was demonstrated with
and ex vivo studies how a short-term treatment with
broad-spectrum antibiotics deeply aﬀected both cellular and humoral immune response [
Some antibiotics have been reported to display immunomodulatory eﬀect in addition to their
antimicrobial activity [
]. Konstantinidis et al. demonstrated that macrolides such as clarithromycin
can induce Neutrophils Extracellular Trap (NET) generation both
in this study the authors showed that clarithromycin-induced NETs are decorated with functional
antimicrobial peptide LL-37, which is able to inhibit the growth of multidrug resistant strains [
In addition, LL-37 plays a critical role in the protection of the colon microbiota balance [
]. Di Fan et
al. found that hypoxia-inducible factor-1
), a transcription factor for human cathelicidin (LL
37), is important for activating innate immune eﬀectors and is the key determinant of Candida albicans
colonization resistance [
]. Moreover, LL-37 plays multiple roles in innate immune responses and
wound healing [
]. Yoshimura et al., in their ex vivo model of CRAMP
mice, showed that
mice developed more severe colitis and succumbed rapidly [
]. Furthermore, Inomata et
al. reported than the antimicrobial peptide LL-37 upregulates the expression of several immune-related
]. The authors investigated the eﬀect of LL-37 on the gene regulation of human gingival
ﬁbroblasts (HGFs). During this study, it was proven that LL-37 alters the expression of 29 genes
that encode TLR-associated proteins. Moreover, LL-37 increased the LPS-upregulated expression of
]. Apart from the well-documented mechanisms related to LL-37 eﬀects on neutrophils and
monocytes, T-cells also respond to LL-37 stimulation via T-cell proliferation, T-cell activation, as well
as the generation of regulatory T-cells (Tregs) [
]. Human antimicrobial peptides are abundantly
secreted by colonic epithelial cells and are critical components of innate immune response against
enteropathogenic bacteria such as Shigella spp., Salmonella spp., and C. diﬃcile. The antibiotics-induced
synthesis of AMPs is the cornerstone mechanism of the indirect action of this group of drugs on the
By the same token, antibiotics’ inﬂuence on intestinal bacterial diversity and long-term abuse has
been identiﬁed as an independent risk factor for metabolic disorder, such as atherosclerosis-driven
events. Kappel et al., in their experimental animal model, showed that the augmented atherosclerosis
induced by antibiotics was correlated to a loss of gut microbiome’s diversity by a reduction in
Bacteroidetes and Clostridia [
]. Moreover, antibiotics as gut microbiome modulators alter the immune
response to various non-infectious diseases and drugs, such as immune checkpoint inhibitors (ICI) in
patients with solid neoplasms. Kapoor et al. reports that the median overall survival of the patients
who received antibiotics in this window was 2.8 months (95% CI: 1.2–4.5) as compared to 9.2 months
(95% CI: 5.2–13.1) in those who did not receive antibiotics p=0.008 .
Antimicrobial agents induce autophagy and the inhibition of the immune response. In this
context, antibiotics may alleviate the progression of the autoimmune and neuroinﬂammatory
]. Studies show that antibiotics may inﬂuence the pathogenesis of neurodegenerative
diseases, such as multiple sclerosis and Amyotrophic Lateral Sclerosis (ALS), through gut microbiome
]. Some antibiotics, such as beta-lactam, except for direct antimicrobial eﬀects also
act as neuromodulators due to the upregulation of the glutamate transporter 1 (GLT-1) expression [
Previous studies have shown that the microbiome plays a critical role in chemotherapy-induced
peripheral neuropathy (CIPN) [
]. Ramakrishna et al. report that chemotherapeutic agents cause
barrier dysfunction, resulting in increased systemic exposure to bacterial products and metabolites,
which promote both local and systemic inﬂammation, which drive pain sensitivity. The authors
Biomedicines 2020,8, 502 6 of 15
believe that microorganisms Porphyromonadaceae are associated with both bacteria and pain as well as
between microglia and pain, and that gut bacteria modiﬁcation by antibiotics has a positive eﬀect on
this phenotype .
Another pathway of the indirect eﬀect of antibiotics on the human microbiome is the regulation of
radical nitric oxide (NO) synthesis by the activation of the inducible nitric oxide synthase. NO increases
mucosal blood ﬂow and mucus thickness and prevents microbial infections [
]. However, the impact
of NO on the gut microbiota remains elusive. Studies indicate that the NO plays a vital role in host
defense against bacterial infections .
Immune cells play a signiﬁcant role in the maintenance of tissue homeostasis by exhibiting the
plasticity of their phenotypes, such as M1 or M2 for macrophages or N1 and N2 for neutrophils.
Microbiota-derived metabolites, short-chain fatty acids (SCFAs), bacterial lipopolysaccharides (LPS),
and antimicrobial peptides wield anti-inﬂammatory or pro-inﬂammatory eﬀects by acting on immune
]. Maekawa et al. demonstrated that the anti-inﬂammatory action of erythromycin is
mediated through the upregulation of the secreted homeostatic protein DEL-1 [
]. Through this
study, it was shown that erythromycin regulates neutrophil function in the tissues, such as lungs or the
periodontium, in a DEL-1-dependent manner (Figure 1).
Biomedicines 2020, 8, x FOR PEER REVIEW 6 of 16
Another pathway of the indirect effect of antibiotics on the human microbiome is the regulation
of radical nitric oxide (NO) synthesis by the activation of the inducible nitric oxide synthase. NO
increases mucosal blood flow and mucus thickness and prevents microbial infections . However,
the impact of NO on the gut microbiota remains elusive. Studies indicate that the NO plays a vital
role in host defense against bacterial infections .
Immune cells play a significant role in the maintenance of tissue homeostasis by exhibiting the
plasticity of their phenotypes, such as M1 or M2 for macrophages or N1 and N2 for neutrophils.
Microbiota-derived metabolites, short-chain fatty acids (SCFAs), bacterial lipopolysaccharides (LPS),
and antimicrobial peptides wield anti-inflammatory or pro-inflammatory effects by acting on
immune cells [103,104]. Maekawa et al. demonstrated that the anti-inflammatory action of
erythromycin is mediated through the upregulation of the secreted homeostatic protein DEL-1 .
Through this study, it was shown that erythromycin regulates neutrophil function in the tissues, such
as lungs or the periodontium, in a DEL-1-dependent manner (Figure 1)
A high-fat diet (HFD) exhibited impaired neutrophil migration to the intestinal mucosa and
reduced the gene expression of the CXCL-1 chemokine and CXCR-2 receptor in the ileum . In
this context, it was previously shown that the depletion of neutrophil migration is also correlated
with the proliferation of tumor cells and tumor-cell DNA damage in an interleukin-17-dependant
manner . Moreover, a high-fat diet induced neutrophil activation by enhancing neutrophil
elastase activity . The high levels of active neutrophil elastase are associated with a low
microbiome diversity and the downregulation of microbiome characteristics . In addition,
neutrophil extracellular traps (NETs), as cornerstone mechanisms of neutrophil action, are involved
in several disease exacerbations. Dicker et al. report that NETs are associated with disease severity in
patients with Chronic Obstructive Pulmonary disease (COPD), a loss of microbiota diversity (p =
0.009), and the dominance of Haemophilus species’ operational taxonomic units (p = 0.01) . Besides
this, it was previously reported than neutrophil ageing is regulated by the microbiome. This
mechanism is driven by the microbiota via the TLR receptor and myeloid differentiation factor 88-
mediated signaling pathways .
Figure 1. Effects of antibiotics upon the gut microbiome. Antibiotic treatment is crucial for combating
infections. On the other hand, antibiotic exposure can alter many basic equilibria in terms of intestinal
Eﬀects of antibiotics upon the gut microbiome. Antibiotic treatment is crucial for
combating infections. On the other hand, antibiotic exposure can alter many basic equilibria
in terms of intestinal microbiota and host immunity, promoting long-term disease. DC: dendritic
cells; DAMP: damage-associated molecular patterns; PMNs: polymorphonuclear leukocytes; PAMP:
pathogen-associated molecular patterns; Th: T helper cells.
A high-fat diet (HFD) exhibited impaired neutrophil migration to the intestinal mucosa and
reduced the gene expression of the CXCL-1 chemokine and CXCR-2 receptor in the ileum [
]. In this
context, it was previously shown that the depletion of neutrophil migration is also correlated with the
proliferation of tumor cells and tumor-cell DNA damage in an interleukin-17-dependant manner [
Moreover, a high-fat diet induced neutrophil activation by enhancing neutrophil elastase activity [
Biomedicines 2020,8, 502 7 of 15
The high levels of active neutrophil elastase are associated with a low microbiome diversity and the
downregulation of microbiome characteristics [
]. In addition, neutrophil extracellular traps (NETs),
as cornerstone mechanisms of neutrophil action, are involved in several disease exacerbations. Dicker
et al. report that NETs are associated with disease severity in patients with Chronic Obstructive
Pulmonary disease (COPD), a loss of microbiota diversity (p=0.009), and the dominance of Haemophilus
species’ operational taxonomic units (p=0.01) [
]. Besides this, it was previously reported than
neutrophil ageing is regulated by the microbiome. This mechanism is driven by the microbiota via the
TLR receptor and myeloid diﬀerentiation factor 88-mediated signaling pathways .
4. The Reservoir of Antibiotics in Animal Feed and the Emerging Resistome
4.1. AR in the Food Chain
Due to the increased development of animal production in industrial plants, antibiotics are added
to feed for the eﬃcient feeding of animals and poultry and for improving their growth. From the
total amount of produced antibiotics, 40% are used for this purpose [
]. In Europe, their use has
been registered since 1953. Penicillin and tetracyclines for low-level feeding (5 to 10 g/ton) are used
in premixes or feed supplements as growth promotions, speciﬁcally in poultry. A plethora of other
antibiotics, such as swine and ruminants, is used for producing meat. In addition to animal growth,
these antibiotics aid in the reduction in enterotoxaemia symptoms. The use of antibiotics for growth
promotion purposes was banned in the European Union in 2006 (European Commission, Brussels
(December 2005): “Ban on antibiotics as growth promoters in animal feed enters into eﬀect”). Similar
actions were taken as of 2017 in the U.S.A. for drugs that are important to human health .
Antibiotics are purchased from the feed industry or from veterinary supply centers and are given
to animals, usually by being placed in their drinking water. In the United States, farmers use more
than 17 antibiotics in animal husbandry [
]. The amount of antibiotics used for infections is four
times less than the quantities used for breeding livestock, as the Food and Drug Administration (FDA)
stated in 2011 [
]. On 18 December 2018, the FDA reported that “domestic sales and distribution
of all medically important antimicrobials intended for use in food-producing animals decreased by
33 percent between the years 2016 and 2017”, probably due to eﬀective antibiotic stewardship .
Antibiotics use also provides a clear beneﬁt for the producer, as less feed is needed for the animal
to achieve the desired weight development; therefore, the cost of purchasing food for the animal is
reduced. Nevertheless, the mechanism of antibiotic use as a growth promoter is not yet clariﬁed.
Animals are believed to develop latent infections following the production of catabolic products and
cytokines that interfere with the growth of animal ﬂesh due to unhygienic conditions during breeding.
Antibiotics can prevent this situation by suppressing pathogens [
]. Alternatively, animal feed
is never sterile. In this vein, bacteria grow by consuming nutrients found in feed and producing
toxic substances that have adverse outcomes on animal health. Therefore, antibiotics overcome these
harmful bacteria in animal feed [
]. However, the use of antibiotics in this way must be banned due
to the increasing problem of AR. The administration of antibiotics leads to the development of AR,
which seems to be associated with the extended use of antibiotics rather than their short-term use [
Therefore, low-level antibiotic feeding causes bacterial resistance [
]. Antibiotics misuse in both
animals and humans leads to a signiﬁcant increase in antibiotic-developed resistance [
], and this
resistance can be transferred through plasmids from resistant bacteria to sensitive ones [
hazards associated with animal health from using low-level antibiotics include the development
of resistant pathogenic strains, as well as increasing susceptibility to several infections due to the
disturbance of the microbiota or to immunosuppression [
]. Promising antimicrobial agents have
been developed and could be used in the animal industry, whilst eﬀective vaccines are available for
enterotoxaemia and other infectious diseases. Should an animal vaccination program be introduced,
the constant demand for disease surveillance through antibiotics could wane, as long as research is
progressing constantly in this direction.
Biomedicines 2020,8, 502 8 of 15
Microbial communities survive in highly antagonistic environments where the nutritional sources
available can deﬁne their growth and genetic persistence. Human activities select resistant strains and
strengthen the transfer of genetic information from unlinked bacterial species by creating environmental
]. Antibiotic resistance is also developed in plant pathogens [
]. Furthermore, domestic,
hospital, and industrial waste contributes to the selection of resistant strains. Thus, resistant bacteria
can be passed onto other hosts in diﬀerent ways, or their mutations can be passed to subsequent
bacterial generations. As stated, environmental niches (pathogenicity islands) that carry multiple
drug-resistant genes can be formed.
Researchers have performed studies in humans under clinical treatment or experimental exposure
(volunteers) to antibiotics [
]. Additionally, other researchers have investigated the diﬀerent functions
of the intestinal microbiota subsequent to antibiotic administration in germ-free animals [
The importance of the ecological equilibrium of the intestine, called “colonization resistance”, as
antibiotic resistance is spreading between humans, should be limited [
]. Apparently, antimicrobials
entered the food chain a long time ago, and human existence has already been continuously inﬂuenced
for a signiﬁcant amount of time. The use of antibiotics as growth promoters is suspected to be
a contributing factor in the emergence of resistant microbial strains responsible for detrimental
4.2. AR Genes in the Intestinal Microbiome
Although the gut microbiome may be considered as the basis of the host’s wellbeing, at the
same time it creates potential threats due to the presence of ARgenes (antibiotic resistance genes).
It could be a “reservoir” of Multi Drug Resistant Bacteria (MDR) or Pan Drug Resistant Bacteria
(PDR) and their antibiotics resistance genes [
]. Taken together, the ARgenes and their ancestors
of pathogenic and non-pathogenic gut bacteria comprise the “resistome”, as proposed by Gerard D.
Wright in 2006 and 2007 [
]. Scattering ARgenes by diﬀerent methods—namely, horizontal gene
transfer, toxin–antitoxin systems, and Mobile Genetic Elements (MGEs)—creates a huge reservoir
of AR determinants in the intestinal microbiome. However, it seems that the majority of these
determinants are considered innate and are not shared with opportunistic pathogens [
the “mobilome”, which consists of MGEs, serves as a path for transferring ARgenes among intestinal
]. Metagenomic research revealed that, after extended antibiotic treatment, especially with
aminoglycosides, an augmentation in the relative abundance of ARgenes emerged [
diﬃcile is a well-known factor causing nosocomial diarrhea because of prolonged broad-spectrum
antibiotic treatment, and it is worth stating that probiotics (beneﬁcial microbes for the gut) together with
antibiotics might prevent clinical infections [
]. Probiotic bacteria as well as dietary interventions
could be very promising, either preventing undesired shifts in the gut microbiome due to antibiotics or
restoring the harmed balance after detrimental antibiotic use [127,134,135].
Antimicrobial resistance poses an immense threat to global health. There is a considerable amount
of evidence from animal models regarding the involvement of disrupted intestinal microbiota under
antibiotic treatment. The role of antibiotics as a catalyst in this interaction, either as therapy or through
low-dosage intake through the food-chain, has not yet been fully clariﬁed. New cutting-edge techniques
and more sophisticated and randomized control trials are required to elucidate the relationship and
examine the potentials and challenges for combating the new epidemic of AR. The World Health
Organization (WHO), following an extended surveillance study of antimicrobial resistance, evinces
the severity of the problem and emphasizes the necessity of concerted action among all states and
involved bodies in order for society to mitigate antimicrobial resistance’s colossal threat. Besides this,
the economic losses linked to antimicrobial resistance should be noted. In this vein, global collaboration
between scientists will permit us to explore and establish the best policy and the most eﬀective process
and strategy in the community and the environment.
Biomedicines 2020,8, 502 9 of 15
Conceptualization, C.T. and E.S.; formal analysis, T.K. and A.K.; investigation, E.S.;
resources, C.V.; writing—original draft preparation, T.K.; writing—review and editing, C.T.; supervision, E.B.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conﬂicts of Interest: The authors declare no conﬂict of interest.
Gaynes, R. The Discovery of Penicillin—New Insights after More Than 75 Years of Clinical Use. Emerg. Infect.
Dis. 2017,23, 849–853. [CrossRef]
Aslam, B.; Wang, W.; Arshad, M.I.; Khurshid, M.; Muzammil, S.; Rasool, M.H.; Nisar, M.A.; Alvi, R.F.;
Aslam, M.A.; Qamar, M.U.; et al. Antibiotic resistance: A rundown of a global crisis. Infect. Drug Resist.
2018,11, 1645–1658. [CrossRef] [PubMed]
de Jong, J.; Bos, J.H.J.; de Vries, T.W.; de Jong-van den Berg, L.T.W. Use of antibiotics in rural and urban
regions in The Netherlands: An observational drug utilization study. BMC Public Health
Russo, V.; Monetti, V.M.; Guerriero, F.; Trama, U.; Guida, A.; Menditto, E.; Orlando, V. Prevalence of
antibiotic prescription in southern Italian outpatients: Real-world data analysis of socioeconomic and
sociodemographic variables at a municipality level. ClinicoEcon. Outcomes Res.
,10, 251–258. [CrossRef]
Stavropoulou, E.; Tsigalou, C.; Bezirtzoglou, E. Spreading of Antimicrobial Resistance (AMR) across clinical
borders. Erciyes Med. J. 2019,41, 238–243. [CrossRef]
Bezirtzoglou, P.E.; Alexopoulos, A.; Voidarou, C. Apparent antibiotic misuse in environmental ecosystems
and food. Microb. Ecol. Health Dis. 2008,20, 197–198. [CrossRef]
Bezirtzoglou, E.; Stavropoulou, E. Immunology and probiotic impact of the newborn and young children
intestinal microﬂora. Anaerobe 2011,17, 369–374. [CrossRef]
Cani, P.D. Gut microbiota—At the intersection of everything? Nat. Rev. Gastroenterol. Hepatol.
Leong, K.S.W.; Derraik, J.G.B.; Hofman, P.L.; Cutﬁeld, W.S. Antibiotics, gut microbiome and obesity.
Clin. Endocrinol. 2018,88, 185–200. [CrossRef]
10. Belizário, J.E.; Faintuch, J. Microbiome and Gut Dysbiosis. Exp. Suppl. 2018,109, 459–476. [CrossRef]
Bartlett, J.G.; Gilbert, D.N.; Spellberg, B. Seven ways to preserve the miracle of antibiotics. Clin. Infect. Dis.
2013,56, 1445–1450. [CrossRef] [PubMed]
Tsigalou, C.; Stavropoulou, E.; Bezirtzoglou, E. Current Insights in Microbiome Shifts in Sjogren’s Syndrome
and Possible Therapeutic Interventions. Front. Immunol. 2018,9. [CrossRef] [PubMed]
rio, J.E.; Napolitano, M. Human microbiomes and their roles in dysbiosis, common diseases, and novel
therapeutic approaches. Front. Microbiol. 2015,6, 1050. [CrossRef]
Sender, R.; Fuchs, S.; Milo, R. Are We Really Vastly Outnumbered? Revisiting the Ratio of Bacterial to Host
Cells in Humans. Cell 2016,164, 337–340. [CrossRef] [PubMed]
Qin, J.; Li, R.; Raes, J.; Arumugam, M.; Burgdorf, K.S.; Manichanh, C.; Nielsen, T.; Pons, N.; Levenez, F.;
Yamada, T.; et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature
2010,464, 59–65. [CrossRef]
Almeida, A.; Mitchell, A.L.; Boland, M.; Forster, S.C.; Gloor, G.B.; Tarkowska, A.; Lawley, T.D.; Finn, R.D.
A new genomic blueprint of the human gut microbiota. Nature 2019,568, 499–504. [CrossRef] [PubMed]
Forster, S.C.; Kumar, N.; Anonye, B.O.; Almeida, A.; Viciani, E.; Stares, M.D.; Dunn, M.; Mkandawire, T.T.;
Zhu, A.; Shao, Y.; et al. A human gut bacterial genome and culture collection for improved metagenomic
analyses. Nat. Biotechnol. 2019,37, 186–192. [CrossRef]
Cani, P.D.; Delzenne, N.M. Gut microﬂora as a target for energy and metabolic homeostasis. Curr. Opin. Clin.
Nutr. Metab. Care 2007,10, 729–734. [CrossRef]
Clarke, G.; Stilling, R.M.; Kennedy, P.J.; Stanton, C.; Cryan, J.F.; Dinan, T.G. Minireview: Gut microbiota:
The neglected endocrine organ. Mol. Endocrinol. 2014,28, 1221–1238. [CrossRef]
Zoetendal, E.G.; Rajilic-Stojanovic, M.; de Vos, W.M. High-throughput diversity and functionality analysis of
the gastrointestinal tract microbiota. Gut 2008,57, 1605–1615. [CrossRef]
Biomedicines 2020,8, 502 10 of 15
Segata, N.; Haake, S.K.; Mannon, P.; Lemon, K.P.; Waldron, L.; Gevers, D.; Huttenhower, C.; Izard, J.
Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat
and stool samples. Genome Biol. 2012,13, R42. [CrossRef] [PubMed]
Arumugam, M.; Raes, J.; Pelletier, E.; Le Paslier, D.; Yamada, T.; Mende, D.R.; Fernandes, G.R.; Tap, J.;
Bruls, T.; Batto, J.-M.; et al. Enterotypes of the human gut microbiome. Nature
,473, 174–180. [CrossRef]
Slingerland, A.E.; Schwabkey, Z.; Wiesnoski, D.H.; Jenq, R.R. Clinical Evidence for the Microbiome in
Inﬂammatory Diseases. Front. Immunol. 2017,8, 400. [CrossRef] [PubMed]
nez, E.; Fern
ndez, L.; Mar
n, M.L.; Mart
n, R.; Odriozola, J.M.; Nueno-Palop, C.; Narbad, A.; Olivares, M.;
Xaus, J.; Rodr
guez, J.M. Isolation of commensal bacteria from umbilical cord blood of healthy neonates born
by cesarean section. Curr. Microbiol. 2005,51, 270–274. [CrossRef]
nez, E.; Mar
n, M.L.; Mart
n, R.; Odriozola, J.M.; Olivares, M.; Xaus, J.; Fern
ndez, L.; Rodr
guez, J.M. Is
meconium from healthy newborns actually sterile? Res. Microbiol. 2008,159, 187–193. [CrossRef]
Aagaard, K.; Ma, J.; Antony, K.M.; Ganu, R.; Petrosino, J.; Versalovic, J. The placenta harbors a unique
microbiome. Sci. Transl. Med. 2014,6, 237ra65. [CrossRef]
Traykova, D.; Schneider, B.; Chojkier, M.; Buck, M. Blood Microbiome Quantity and the Hyperdynamic
Circulation in Decompensated Cirrhotic Patients. PLoS ONE 2017,12, e0169310. [CrossRef]
28. Neuman, H.; Forsythe, P.; Uzan, A.; Avni, O.; Koren, O. Antibiotics in early life: Dysbiosis and the damage
done. FEMS Microbiol. Rev. 2018,42, 489–499. [CrossRef]
De Filippo, C.; Cavalieri, D.; Di Paola, M.; Ramazzotti, M.; Poullet, J.B.; Massart, S.; Collini, S.; Pieraccini, G.;
Lionetti, P. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe
and rural Africa. Proc. Natl. Acad. Sci. USA 2010,107, 14691–14696. [CrossRef]
Bezirtzoglou, E.; Romond, C. Occurrence of Biﬁdobacterium in the feces of newborns delivered by cesarean
section. Biol. Neonate 1990,58, 247–251. [CrossRef]
Mitsuoka, T.; Hayakawa, K. The fecal ﬂora in man. I. Composition of the fecal ﬂora of various age groups.
Zentralbl. Bakteriol. Orig. A 1973,223, 333–342. [PubMed]
Ellis-Pegler, R.B.; Crabtree, C.; Lambert, H.P. The faecal ﬂora of children in the United Kingdom. J. Hyg.
1975,75, 135–142. [CrossRef] [PubMed]
Zetterström, R.; Bennet, R.; Eriksson, M. Sepsis in newborn infants: Its incidence, etiology and prognosis.
Pediatriia 1988, 36–40.
Hentges, D.J. Human Intestinal Microﬂora in Health and Disease; Academic Press: Cambridge, MA, USA, 1983;
Koenig, J.E.; Spor, A.; Scalfone, N.; Fricker, A.D.; Stombaugh, J.; Knight, R.; Angenent, L.T.; Ley, R.E.
Succession of microbial consortia in the developing infant gut microbiome. Proc. Natl. Acad. Sci. USA
108 (Suppl. 1), 4578–4585. [CrossRef]
Bezirtzoglou, E.; Romond, M.B.; Romond, C. Modulation of Clostridium perfringens intestinal colonization
in infants delivered by caesarean section. Infection 1989,17, 232–236. [CrossRef]
Salminen, S.; von Wright, A. Lactic Acid Bacteria: Microbiology and Functional Aspects, 2nd ed.; Marcel Dekker:
New York, NY, USA, 1998; ISBN 978-0-8247-0133-8.
Gaon, D.; Garmendia, C.; Murrielo, N.O.; de Cucco Games, A.; Cerchio, A.; Quintas, R.; Gonz
Oliver, G. Eﬀect of Lactobacillus strains (L. casei and L. acidophillus Strains cerela) on bacterial
overgrowth-related chronic diarrhea. Medicina 2002,62, 159–163.
Chakraborti, C.K. New-found link between microbiota and obesity. World J. Gastrointest. Pathophysiol.
6, 110–119. [CrossRef]
Van Tyne, D.; Manson, A.L.; Huycke, M.M.; Karanicolas, J.; Earl, A.M.; Gilmore, M.S. Impact of antibiotic
treatment and host innate immune pressure on enterococcal adaptation in the human bloodstream. Sci. Transl.
Med. 2019,11. [CrossRef]
Murphy, E.F.; Cotter, P.D.; Healy, S.; Marques, T.M.; O’Sullivan, O.; Fouhy, F.; Clarke, S.F.; O’Toole, P.W.;
Quigley, E.M.; Stanton, C.; et al. Composition and energy harvesting capacity of the gut microbiota:
Relationship to diet, obesity and time in mouse models. Gut 2010,59, 1635–1642. [CrossRef]
Palmer, C.; Bik, E.M.; DiGiulio, D.B.; Relman, D.A.; Brown, P.O. Development of the human infant intestinal
microbiota. PLoS Biol. 2007,5, e177. [CrossRef]
Biomedicines 2020,8, 502 11 of 15
Nord, C.E.; Edlund, C. Impact of antimicrobial agents on human intestinal microﬂora. J. Chemother.
218–237. [CrossRef] [PubMed]
Agerholm-Larsen, L.; Raben, A.; Haulrik, N.; Hansen, A.S.; Manders, M.; Astrup, A. Eﬀect of 8 week intake
of probiotic milk products on risk factors for cardiovascular diseases. Eur. J. Clin. Nutr.
Crovesy, L.; Ostrowski, M.; Ferreira, D.M.T.P.; Rosado, E.L.; Soares-Mota, M. Eﬀect of Lactobacillus on body
weight and body fat in overweight subjects: A systematic review of randomized controlled clinical trials.
Int. J. Obes. 2017,41, 1607–1614. [CrossRef] [PubMed]
Rolhion, N.; Chassaing, B. When pathogenic bacteria meet the intestinal microbiota. Philos. Trans. R. Soc.
Lond. B Biol. Sci. 2016,371. [CrossRef] [PubMed]
Mikelsaar, M. Human microbial ecology: Lactobacilli, probiotics, selective decontamination. Anaerobe
17, 463–467. [CrossRef]
Sequeira, S.; Kavanaugh, D.; MacKenzie, D.A.; Šuligoj, T.; Walpole, S.; Leclaire, C.; Gunning, A.P.;
Latousakis, D.; Willats, W.G.T.; Angulo, J.; et al. Structural basis for the role of serine-rich repeat proteins from
Lactobacillus reuteri in gut microbe–host interactions. Proc. Natl. Acad. Sci. USA
Ingrassia, I.; Leplingard, A.; Darfeuille-Michaud, A. Lactobacillus casei DN-114 001 Inhibits the Ability of
Adherent-Invasive Escherichia coli Isolated from Crohn’s Disease Patients To Adhere to and To Invade
Intestinal Epithelial Cells. Appl. Environ. Microbiol. 2005,71, 2880–2887. [CrossRef]
Jayasinghe, T.N.; Chiavaroli, V.; Holland, D.J.; Cutﬁeld, W.S.; O’Sullivan, J.M. The New Era of Treatment
for Obesity and Metabolic Disorders: Evidence and Expectations for Gut Microbiome Transplantation.
Front. Cell. Infect. Microbiol. 2016,6, 15. [CrossRef]
Walker, A.W.; Ince, J.; Duncan, S.H.; Webster, L.M.; Holtrop, G.; Ze, X.; Brown, D.; Stares, M.D.; Scott, P.;
Bergerat, A.; et al. Dominant and diet-responsive groups of bacteria within the human colonic microbiota.
ISME J. 2011,5, 220–230. [CrossRef]
Binns, N. Probiotics, Prebiotics and the Gut Microbiota; ILSI Europe: Brussels, Belgium, 2013; ISBN 978-90-78637-39-4.
Modi, S.R.; Collins, J.J.; Relman, D.A. Antibiotics and the gut microbiota. J. Clin. Investig.
Mutlu, E.A.; Gillevet, P.M.; Rangwala, H.; Sikaroodi, M.; Naqvi, A.; Engen, P.A.; Kwasny, M.; Lau, C.K.;
Keshavarzian, A. Colonic microbiome is altered in alcoholism. Am. J. Physiol. Gastrointest. Liver Physiol.
2012,302, G966–G978. [CrossRef] [PubMed]
Davies, J.; Davies, D. Origins and evolution of antibiotic resistance. Microbiol. Mol. Biol. Rev.
417–433. [CrossRef] [PubMed]
Smith, R.A.; M’ikanatha, N.M.; Read, A.F. Antibiotic resistance: A primer and call to action. Health Commun.
2015,30, 309–314. [CrossRef] [PubMed]
Vrieze, A.; Out, C.; Fuentes, S.; Jonker, L.; Reuling, I.; Kootte, R.S.; van Nood, E.; Holleman, F.; Knaapen, M.;
Romijn, J.A.; et al. Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity.
J. Hepatol. 2014,60, 824–831. [CrossRef] [PubMed]
58. Jernberg, C.; Löfmark, S.; Edlund, C.; Jansson, J.K. Long-term impacts of antibiotic exposure on the human
intestinal microbiota. Microbiology 2010,156, 3216–3223. [CrossRef]
Panda, S.; El khader, I.; Casellas, F.; L
pez Vivancos, J.; Garc
a Cors, M.; Santiago, A.; Cuenca, S.; Guarner, F.;
Manichanh, C. Short-term eﬀect of antibiotics on human gut microbiota. PLoS ONE
,9, e95476. [CrossRef]
Zaura, E.; Brandt, B.W.; Teixeira de Mattos, M.J.; Buijs, M.J.; Caspers, M.P.M.; Rashid, M.-U.; Weintraub, A.;
Nord, C.E.; Savell, A.; Hu, Y.; et al. Same Exposure but Two Radically Diﬀerent Responses to Antibiotics:
Resilience of the Salivary Microbiome versus Long-Term Microbial Shifts in Feces. MBio
Yassour, M.; Vatanen, T.; Siljander, H.; Hämäläinen, A.-M.; Härkönen, T.; Ryhänen, S.J.; Franzosa, E.A.;
Vlamakis, H.; Huttenhower, C.; Gevers, D.; et al. Natural history of the infant gut microbiome and impact of
antibiotic treatment on bacterial strain diversity and stability. Sci. Transl. Med. 2016,8, 343ra81. [CrossRef]
de Lastours, V.; Fantin, B. R
sistance aux ﬂuoroquinolones en 2010: Quel impact pour la prescription en
réanimation ? Réanimation 2010,19, 347–353. [CrossRef]
Biomedicines 2020,8, 502 12 of 15
Johanesen, P.A.; Mackin, K.E.; Hutton, M.L.; Awad, M.M.; Larcombe, S.; Amy, J.M.; Lyras, D. Disruption of
the Gut Microbiome: Clostridium diﬃcile Infection and the Threat of Antibiotic Resistance. Genes
Leﬄer, D.A.; Lamont, J.T. Clostridium diﬃcile Infection. N. Engl. J. Med.
,373, 287–288. [CrossRef]
Belkaid, Y.; Hand, T.W. Role of the microbiota in immunity and inﬂammation. Cell
Cox, L.M.; Blaser, M.J. Antibiotics in early life and obesity. Nat. Rev. Endocrinol.
,11, 182–190. [CrossRef]
Arat, S.; Spivak, A.; Van Horn, S.; Thomas, E.; Traini, C.; Sathe, G.; Livi, G.P.; Ingraham, K.; Jones, L.;
Aubart, K.; et al. Microbiome changes in healthy volunteers treated with GSK1322322, a novel antibiotic
targeting bacterial peptide deformylase. Antimicrob. Agents Chemother.
,59, 1182–1192. [CrossRef]
Arboleya, S.; S
nchez, B.; Milani, C.; Duranti, S.; Sol
s, G.; Fern
ndez, N.; de los Reyes-Gavil
Ventura, M.; Margolles, A.; Gueimonde, M. Intestinal microbiota development in preterm neonates and eﬀect
of perinatal antibiotics. J. Pediatr. 2015,166, 538–544. [CrossRef] [PubMed]
Clarke, S.F.; Murphy, E.F.; O’Sullivan, O.; Lucey, A.J.; Humphreys, M.; Hogan, A.; Hayes, P.; O’Reilly, M.;
Jeﬀery, I.B.; Wood-Martin, R.; et al. Exercise and associated dietary extremes impact on gut microbial diversity.
Gut 2014,63, 1913–1920. [CrossRef]
Abdulkadir, B.; Nelson, A.; Skeath, T.; Marrs, E.C.L.; Perry, J.D.; Cummings, S.P.; Embleton, N.D.;
Berrington, J.E.; Stewart, C.J. Routine Use of Probiotics in Preterm Infants: Longitudinal Impact on
the Microbiome and Metabolome. Neonatology 2016,109, 239–247. [CrossRef]
Cox, L.M.; Yamanishi, S.; Sohn, J.; Alekseyenko, A.V.; Leung, J.M.; Cho, I.; Kim, S.G.; Li, H.; Gao, Z.;
Mahana, D.; et al. Altering the intestinal microbiota during a critical developmental window has lasting
metabolic consequences. Cell 2014,158, 705–721. [CrossRef]
Bezirtzoglou, E.E.V. Intestinal cytochromes P450 regulating the intestinal microbiota and its probiotic proﬁle.
Microb. Ecol. Health Dis. 2012,23. [CrossRef]
Korpela, K.; de Vos, W.M. Early life colonization of the human gut: Microbes matter everywhere. Curr. Opin.
Microbiol. 2018,44, 70–78. [CrossRef]
Edwards, A.N.; Karim, S.T.; Pascual, R.A.; Jowhar, L.M.; Anderson, S.E.; McBride, S.M. Chemical and Stress
Resistances of Clostridium diﬃcile Spores and Vegetative Cells. Front. Microbiol.
,7, 1698. [CrossRef]
Korpela, K.; de Vos, W.M. Antibiotic use in childhood alters the gut microbiota and predisposes to overweight.
Microb Cell 2016,3, 296–298. [CrossRef] [PubMed]
Drummond, L.J.; Smith, D.G.E.; Poxton, I.R. Eﬀects of sub-MIC concentrations of antibiotics on growth of
and toxin production by Clostridium diﬃcile. J. Med. Microbiol. 2003,52, 1033–1038. [CrossRef] [PubMed]
Zhernakova, A.; Kurilshikov, A.; Bonder, M.J.; Tigchelaar, E.F.; Schirmer, M.; Vatanen, T.; Mujagic, Z.;
Vila, A.V.; Falony, G.; Vieira-Silva, S.; et al. Population-based metagenomics analysis reveals markers for gut
microbiome composition and diversity. Science 2016,352, 565–569. [CrossRef]
Amoroso, C.; Perillo, F.; Strati, F.; Fantini, M.; Caprioli, F.; Facciotti, F. The Role of Gut Microbiota
Biomodulators on Mucosal Immunity and Intestinal Inﬂammation. Cells 2020,9. [CrossRef]
Shuang, G.; Yu, S.; Weixiao, G.; Dacheng, W.; Zhichao, Z.; Jing, L.; Xuming, D. Immunosuppressive activity
of ﬂorfenicol on the immune responses in mice. Immunol. Investig. 2011,40, 356–366. [CrossRef]
Cho, I.; Yamanishi, S.; Cox, L.; Meth
, B.A.; Zavadil, J.; Li, K.; Gao, Z.; Mahana, D.; Raju, K.; Teitler, I.;
et al. Antibiotics in early life alter the murine colonic microbiome and adiposity. Nature
Grochla, I.; Ko, H.L.; Beuth, J.; Roszkowski, K.; Roszkowski, W.; Pulverer, G. Eﬀects of beta-lactam antibiotics
imipenem/cilastatin and cefodizime on cellular and humoral immune responses in BALB/c-mice. Zentralbl.
Bakteriol. 1990,274, 250–258. [CrossRef]
Garrido-Mesa, N.; Camuesco, D.; Arribas, B.; Comalada, M.; Bail
n, E.; Cueto-Sola, M.; Utrilla, P.; Nieto, A.;
Zarzuelo, A.; Rodr
guez-Cabezas, M.E.; et al. The intestinal anti-inﬂammatory eﬀect of minocycline in
experimental colitis involves both its immunomodulatory and antimicrobial properties. Pharmacol. Res.
2011,63, 308–319. [CrossRef]
Biomedicines 2020,8, 502 13 of 15
Garrido-Mesa, J.; Rodr
guez-Nogales, A.; Algieri, F.; Vezza, T.; Hidalgo-Garcia, L.; Garrido-Barros, M.;
Utrilla, M.P.; Garcia, F.; Chueca, N.; Rodriguez-Cabezas, M.E.; et al. Immunomodulatory tetracyclines shape
the intestinal inﬂammatory response inducing mucosal healing and resolution. Br. J. Pharmacol.
Konstantinidis, T.; Kambas, K.; Mitsios, A.; Panopoulou, M.; Tsironidou, V.; Dellaporta, E.; Kouklakis, G.;
Arampatzioglou, A.; Angelidou, I.; Mitroulis, I.; et al. Immunomodulatory Role of Clarithromycin in
Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps. Antimicrob. Agents
Chemother. 2016,60, 1040–1048. [CrossRef] [PubMed]
Zhang, M.; Liang, W.; Gong, W.; Yoshimura, T.; Chen, K.; Wang, J.M. The Critical Role of the Antimicrobial
Peptide LL-37/CRAMP in Protection of Colon Microbiota Balance, Mucosal Homeostasis, Anti-Inﬂammatory
Responses, and Resistance to Carcinogenesis. Crit. Rev. Immunol. 2019,39, 83–92. [CrossRef] [PubMed]
Fan, D.; Coughlin, L.A.; Neubauer, M.M.; Kim, J.; Kim, M.S.; Zhan, X.; Simms-Waldrip, T.R.; Xie, Y.;
Hooper, L.V.; Koh, A.Y. Activation of HIF-1
and LL-37 by commensal bacteria inhibits Candida albicans
colonization. Nat. Med. 2015,21, 808–814. [CrossRef] [PubMed]
Yoshimura, T.; McLean, M.H.; Dzutsev, A.K.; Yao, X.; Chen, K.; Huang, J.; Gong, W.; Zhou, J.; Xiang, Y.; H
Badger, J.; et al. The Antimicrobial Peptide CRAMP Is Essential for Colon Homeostasis by Maintaining
Microbiota Balance. J. Immunol. 2018,200, 2174–2185. [CrossRef]
Arampatzioglou, A.; Papazoglou, D.; Konstantinidis, T.; Chrysanthopoulou, A.; Mitsios, A.; Angelidou, I.;
Maroulakou, I.; Ritis, K.; Skendros, P. Clarithromycin Enhances the Antibacterial Activity and Wound
Healing Capacity in Type 2 Diabetes Mellitus by Increasing LL-37 Load on Neutrophil Extracellular Traps.
Front. Immunol. 2018,9, 2064. [CrossRef]
Inomata, M.; Horie, T.; Into, T. Eﬀect of the Antimicrobial Peptide LL-37 on Gene Expression of Chemokines
and 29 Toll-like Receptor-Associated Proteins in Human Gingival Fibroblasts Under Stimulation with
Porphyromonas gingivalis Lipopolysaccharide. Probiotics Antimicrob. Proteins 2020,12, 64–72. [CrossRef]
Alexandre-Ramos, D.S.; Silva-Carvalho, A.
.; Lacerda, M.G.; Serejo, T.R.T.; Franco, O.L.; Pereira, R.W.;
Carvalho, J.L.; Neves, F.A.R.; Saldanha-Araujo, F. LL-37 treatment on human peripheral blood mononuclear
cells modulates immune response and promotes regulatory T-cells generation. Biomed. Pharmacother.
108, 1584–1590. [CrossRef]
Kappel, B.A.; De Angelis, L.; Heiser, M.; Ballanti, M.; Stoehr, R.; Goettsch, C.; Mavilio, M.; Artati, A.;
Paoluzi, O.A.; Adamski, J.; et al. Cross-omics analysis revealed gut microbiome-related metabolic pathways
underlying atherosclerosis development after antibiotics treatment. Mol. Metab.
,36, 100976. [CrossRef]
Kapoor, A.; Noronha, V.; Patil, V.M.; Joshi, A.; Menon, N.; Mahajan, A.; Janu, A.; Prabhash, K. Concomitant
use of antibiotics and immune checkpoint inhibitors in patients with solid neoplasms: Retrospective data
from real-world settings. Ecancermedicalscience 2020,14, 1038. [CrossRef]
Xu, L.; Zhang, C.; He, D.; Jiang, N.; Bai, Y.; Xin, Y. Rapamycin and MCC950 modiﬁed gut microbiota in
experimental autoimmune encephalomyelitis mouse by brain gut axis. Life Sci.
,253, 117747. [CrossRef]
Hill, J.M.; Clement, C.; Pogue, A.I.; Bhattacharjee, S.; Zhao, Y.; Lukiw, W.J. Pathogenic microbes, the
microbiome, and Alzheimer’s disease (AD). Front. Aging Neurosci. 2014,6, 127. [CrossRef] [PubMed]
Obrenovich, M.; Jaworski, H.; Tadimalla, T.; Mistry, A.; Sykes, L.; Perry, G.; Bonomo, R.A. The Role of the
Microbiota-Gut-Brain Axis and Antibiotics in ALS and Neurodegenerative Diseases. Microorganisms
Sasmita, A.O. Modiﬁcation of the gut microbiome to combat neurodegeneration. Rev. Neurosci.
795–805. [CrossRef] [PubMed]
Zumkehr,J.; Rodriguez-Ortiz, C.J.; Cheng, D.; Kieu, Z.; Wai, T.; Hawkins, C.; Kilian, J.; Lim,S.L.; Medeiros, R.;
Kitazawa, M. Ceftriaxone ameliorates tau pathology and cognitive decline via restoration of glial glutamate
transporter in a mouse model of Alzheimer’s disease. Neurobiol. Aging
,36, 2260–2271. [CrossRef]
Zhong, S.; Zhou, Z.; Liang, Y.; Cheng, X.; Li, Y.; Teng, W.; Zhao, M.; Liu, C.; Guan, M.; Zhao, C. Targeting
strategies for chemotherapy-induced peripheral neuropathy: Does gut microbiota play a role? Crit. Rev.
Microbiol. 2019,45, 369–393. [CrossRef]
Bajic, J.E.; Johnston, I.N.; Howarth, G.S.; Hutchinson, M.R. From the Bottom-Up: Chemotherapy and
Gut-Brain Axis Dysregulation. Front. Behav. Neurosci. 2018,12, 104. [CrossRef]
Biomedicines 2020,8, 502 14 of 15
Ramakrishna, C.; Corleto, J.; Ruegger, P.M.; Logan, G.D.; Peacock, B.B.; Mendonca, S.; Yamaki, S.; Adamson, T.;
Ermel, R.; McKemy, D.; et al. Dominant Role of the Gut Microbiota in Chemotherapy Induced Neuropathic
Pain. Sci. Rep. 2019,9, 20324. [CrossRef]
Rocha, B.S.; Correia, M.G.; Pereira, A.; Henriques, I.; Da Silva, G.J.; Laranjinha, J. Inorganic nitrate prevents
the loss of tight junction proteins and modulates inﬂammatory events induced by broad-spectrum antibiotics:
A role for intestinal microbiota? Nitric Oxide 2019,88, 27–34. [CrossRef]
Svensson, L.; Poljakovic, M.; Demirel, I.; Sahlberg, C.; Persson, K. Host-Derived Nitric Oxide and Its
Antibacterial Eﬀects in the Urinary Tract. Adv. Microb. Physiol. 2018,73, 1–62. [CrossRef]
Giraud-Gatineau, A.; Coya, J.M.; Maure, A.; Biton, A.; Thomson, M.; Bernard, E.M.; Marrec, J.; Gutierrez, M.G.;
Larrouy-Maumus, G.; Brosch, R.; et al. The antibiotic bedaquiline activates host macrophage innate immune
resistance to bacterial infection. Elife 2020,9. [CrossRef]
Wang, J.; Chen, W.-D.; Wang, Y.-D. The Relationship Between Gut Microbiota and Inﬂammatory Diseases:
The Role of Macrophages. Front. Microbiol. 2020,11, 1065. [CrossRef] [PubMed]
Maekawa, T.; Tamura, H.; Domon, H.; Hiyoshi, T.; Isono, T.; Yonezawa, D.; Hayashi, N.; Takahashi, N.;
Tabeta, K.; Maeda, T.; et al. Erythromycin inhibits neutrophilic inﬂammation and mucosal disease by
upregulating DEL-1. JCI Insight 2020. [CrossRef] [PubMed]
rez, M.M.; Martins, L.M.S.; Dias, M.S.; Pereira, C.A.; Leite, J.A.; Gonçalves, E.C.S.; de Almeida, P.Z.; de
Freitas, E.N.; Tostes, R.C.; Ramos, S.G.; et al. Interleukin-17/interleukin-17 receptor axis elicits intestinal
neutrophil migration, restrains gut dysbiosis and lipopolysaccharide translocation in high-fat diet-induced
metabolic syndrome model. Immunology 2019,156, 339–355. [CrossRef] [PubMed]
Triner, D.; Devenport, S.N.; Ramakrishnan, S.K.; Ma, X.; Frieler, R.A.; Greenson, J.K.; Inohara, N.; Nunez, G.;
Colacino, J.A.; Mortensen, R.M.; et al. Neutrophils Restrict Tumor-Associated Microbiota to Reduce Growth
and Invasion of Colon Tumors in Mice. Gastroenterology 2019,156, 1467–1482. [CrossRef]
Motoyama, S.; Yamada, H.; Yamamoto, K.; Wakana, N.; Terada, K.; Kikai, M.; Wada, N.; Saburi, M.;
Sugimoto, T.; Kubota, H.; et al. Social Stress Increases Vulnerability to High-Fat Diet-Induced Insulin
Resistance by Enhancing Neutrophil Elastase Activity in Adipose Tissue. Cells 2020,9. [CrossRef]
Oriano, M.; Gramegna, A.; Terranova, L.; Sotgiu, G.; Sulaiman, I.; Ruggiero, L.; Saderi, L.; Wu, B.;
Chalmers, J.D.; Segal, L.N.; et al. Sputum Neutrophil Elastase associates with microbiota and P. aeruginosa
in bronchiectasis. Eur. Respir. J. 2020. [CrossRef]
Dicker, A.J.; Crichton, M.L.; Pumphrey, E.G.; Cassidy, A.J.; Suarez-Cuartin, G.; Sibila, O.; Furrie, E.; Fong, C.J.;
Ibrahim, W.; Brady, G.; et al. Neutrophil extracellular traps are associated with disease severity and microbiota
diversity in patients with chronic obstructive pulmonary disease. J. Allergy Clin. Immunol.
Zhang, D.; Chen, G.; Manwani, D.; Mortha, A.; Xu, C.; Faith, J.J.; Burk, R.D.; Kunisaki, Y.; Jang, J.-E.;
Scheiermann, C.; et al. Neutrophil ageing is regulated by the microbiome. Nature
National Research Council (US). Committee to Study the Human Health Eﬀects of Subtherapeutic Antibiotic
Use in Animal Feeds. In Antibiotics in Animal Feeds; National Academies Press: Washington, DC, USA, 1980.
CDC Antibiotic Resistance and Food Are Connected. Available online: https://www.cdc.gov/drugresistance/
food.html (accessed on 27 October 2020).
Anderson, A.D.; Nelson, J.M.; Rossiter, S.; Angulo, F.J. Public health consequences of use of antimicrobial
agents in food animals in the United States. Microb. Drug Resist. 2003,9, 373–379. [CrossRef]
U.S. Food & Drug Administration. 2017 Summary Report on Antimicrobials Sold or Distributed for Use in
Food-Producing Animals; U.S. Food & Drug Administration: Tulsa, OK, USA, 2018; 52p.
FDA, US. FDA Releases Annual Summary Report on Antimicrobials Sold or Distributed in 2017 for Use in
Food-Producing Animals Showing Declines for Past Two Years; FDA: Tulsa, OK, USA, 2020.
Chattopadhyay, M.K. Use of antibiotics as feed additives: A burning question. Front. Microbiol.
Linton, A.H.; Howe, K.; Bennett, P.M.; Richmond, M.H.; Whiteside, E.J. The Colonization of the Human
Gut by Antibiotic Resistant Escherichia coli from Chickens. J. Appl. Bacteriol.
,43, 465–469. [CrossRef]
Richmond, M.H.; Linton, K.B. The use of tetracycline in the community and its possible relation to the
excretion of tetracycline-resistant bacteria. J. Antimicrob. Chemother. 1980,6, 33–41. [CrossRef] [PubMed]
Biomedicines 2020,8, 502 15 of 15
Septimus, E.J. Antimicrobial Resistance: An Antimicrobial/Diagnostic Stewardship and Infection Prevention
Approach. Med. Clin. N. Am. 2018,102, 819–829. [CrossRef] [PubMed]
Teoh, L.; Stewart, K.; Marino, R.; McCullough, M. Antibiotic resistance and relevance to general dental
practice in Australia. Aust. Dent. J. 2018,63, 414–421. [CrossRef]
Kruse, H.; Sørum, H. Transfer of multiple drug resistance plasmids between bacteria of diverse origins in
natural microenvironments. Appl. Environ. Microbiol. 1994,60, 4015–4021. [CrossRef]
Bauer, M.A.; Kainz, K.; Carmona-Gutierrez, D.; Madeo, F. Microbial wars: Competition in ecological niches
and within the microbiome. Microb. Cell 2018,5, 215–219. [CrossRef]
Sundin, G.W.; Wang, N. Antibiotic Resistance in Plant-Pathogenic Bacteria. Annu. Rev. Phytopathol.
Midtvedt, T.; Lingaas, E.; Carlstedt-Duke, B.; Höverstad, T.; Midtvedt, A.C.; Saxerholt, H.; Steinbakk, M.;
Norin, K.E. Intestinal microbial conversion of cholesterol to coprostanol in man. Inﬂuence of antibiotics.
APMIS 1990,98, 839–844. [CrossRef]
Sullivan, A.; Edlund, C.; Nord, C.E. Eﬀect of antimicrobial agents on the ecological balance of human
microﬂora. Lancet Infect. Dis. 2001,1, 101–114. [CrossRef]
Tsigalou, C.; Konstantinidis, T.; Stavropoulou, E.; Bezirtzoglou, E.E.; Tsakris, A. Potential Elimination of
Human Gut Resistome by Exploiting the Beneﬁts of Functional Foods. Front. Microbiol.
Wright, G.D. The antibiotic resistome: The nexus of chemical and genetic diversity. Nat. Rev. Microbiol.
5, 175–186. [CrossRef] [PubMed]
D’Costa, V.M.; McGrann, K.M.; Hughes, D.W.; Wright, G.D. Sampling the Antibiotic Resistome. Science
311, 374–377. [CrossRef] [PubMed]
, E.; Ghozlane, A.; Tap, J.; Pons, N.; Alvarez, A.-S.; Maziers, N.; Cuesta, T.; Hernando-Amado, S.;
Clares, I.; Mart
nez, J.L.; et al. Prediction of the intestinal resistome by a three-dimensional structure-based
method. Nat. Microbiol. 2019,4, 112–123. [CrossRef] [PubMed]
de Smet, A.M.G.A.; Kluytmans, J.A.J.W.; Cooper, B.S.; Mascini, E.M.; Benus, R.F.J.; van der Werf, T.S.; van der
Hoeven, J.G.; Pickkers, P.; Bogaers-Hofman, D.; van der Meer, N.J.M.; et al. Decontamination of the Digestive
Tract and Oropharynx in ICU Patients. N. Engl. J. Med. 2009,360, 20–31. [CrossRef]
Buelow, E.; Gonzalez, T.B.; Versluis, D.; Oostdijk, E.A.N.; Ogilvie, L.A.; van Mourik, M.S.M.; Oosterink, E.;
van Passel, M.W.J.; Smidt, H.; D’Andrea, M.M.; et al. Eﬀects of selective digestive decontamination (SDD) on
the gut resistome. J. Antimicrob. Chemother. 2014,69, 2215–2223. [CrossRef]
Goldenberg, J.Z.; Mertz, D.; Johnston, B.C. Probiotics to Prevent Clostridium diﬃcile Infection in Patients
Receiving Antibiotics. JAMA 2018,320, 499. [CrossRef]
Wu, G.; Zhang, C.; Wang, J.; Zhang, F.; Wang, R.; Shen, J.; Wang, L.; Pang, X.; Zhang, X.; Zhao, L.; et al.
Diminution of the gut resistome after a gut microbiota-targeted dietary intervention in obese children.
Sci. Rep. 2016,6, 24030. [CrossRef]
Zhang, C.; Yin, A.; Li, H.; Wang, R.; Wu, G.; Shen, J.; Zhang, M.; Wang, L.; Hou, Y.; Ouyang, H.; et al. Dietary
Modulation of Gut Microbiota Contributes to Alleviation of Both Genetic and Simple Obesity in Children.
EBioMedicine 2015,2, 968–984. [CrossRef]
MDPI stays neutral with regard to jurisdictional claims in published maps and institutional
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).