Effect of antibiotics on gut microbiota, glucose metabolism and bodyweight

regulation - a review of the literature

Accepted Article
Kristian Hallundbæk Mikkelsen1, 2, Kristine Højgaard Allin2, Filip Krag Knop1, 2

1) Center for Diabetes Research, Gentofte Hospital, University of Copenhagen, Hellerup, Denmark

2) The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen,

Copenhagen, Denmark

Abbreviated title: Antibiotics, microbiota and metabolism - a review

Corresponding author and person to who reprint requests should be addressed:

Kristian Hallundbæk Mikkelsen, Center for Diabetes Research, Gentofte University Hospital, Kildegårdsvej

28, 2900 Hellerup, Denmark.

E-mail: kristianmikkel@gmail.com

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1002/dom.12637

This article is protected by copyright. All rights reserved.

 Abstract

Gut bacteria are involved in a number of host metabolic processes and have been implicated in the

development of obesity and type 2 diabetes in humans.

Accepted Article
Use of antibiotics changes the composition of the gut microbiota and there is accumulating evidence from

observational studies for an association between exposure to antibiotics and development of obesity and type

2 diabetes.

Here we review human studies examining effects of antibiotics on bodyweight regulation and glucose

metabolism and discuss whether the observed findings may relate to alterations in the composition and

function of the gut microbiota.

This article is protected by copyright. All rights reserved.

 Introduction

The human gut is populated by a dense community of microbes, the gut microbiota, that many-fold

outnumbers our eukaryotic cell count and provides the host with an enormous complimentary microbial gene
Accepted Article
set, the gut microbiome [1]. In cross-sectional studies, an altered gut microbiota has been found in

individuals with obesity and type 2 diabetes compared with lean and glucose-tolerant controls, respectively

[2–4]. Concurrently, intervention studies using fecal microbiota transplantation, have provided evidence for

a causal role of the gut microbiota in the regulation of glucose and lipid metabolism [5–8]. Antibiotics have

well-defined effects on specific infectious bacteria, while effects of antibiotics on the gut microbial

community and the human metabolism have only recently been explored.

This review provides an overview of the effects of antibiotics on bodyweight regulation and glucose

metabolism and discusses whether this may relate to alterations in the composition and function of the gut

microbiota. In the first two sections, we review human observational and clinical studies examining the

effect of antibiotics on bodyweight homeostasis and glucose metabolism. In the third section, we describe

the effects of antibiotics on gut microbial ecology and in the last section we evaluate molecular pathways

linking antibiotics, gut microbial changes and bodyweight homeostasis and glucose metabolism.

Effects of antibiotics on bodyweight

The idea that antibiotics may affect bodyweight is not new. Shortly after the discovery of penicillin it was

noted that adding antibiotics to the feed of farm animals could enhance growth rate and increase food

utilization of the animals [9]. Furthermore, this effect was replicated with several types of antibiotics and in a

number of animal species as previously reviewed [10]. In humans, the effect of antibiotics on bodyweight

has been studied in both children and adults, but with slightly different methodologies. In the first part of this

section, we will first review the data from the predominantly register-based studies of children and infants.

This article is protected by copyright. All rights reserved.

 Studies in children

A number of interventional studies have examined the effect of antibiotics on bodyweight in children, but

these studies have included only children with concomitant infections or children at risk of severe infections.
Accepted Article
As examples, weight gain and increased body mass index (BMI) has been reported following prophylactic

antibiotics treatment of children with cystic fibrosis [11] and a meta-analysis including 10 randomized

controlled trials, found increased growth rate with antibiotics vs placebo in children with malnutrition,

diarrhea or amoebiasis [12]. The findings of weight gain were in many of these studies considered as a

benefit, and it seems difficult to separate the effect of treating parasitic or subclinical infections from other

antibiotic-mediated effects in these studies. We will therefore focus on the more recent and alarming results

from observational studies of unselected or otherwise healthy children.

Starting already before birth, two recent observational studies from the US and Denmark, respectively,

suggested that maternal antibiotic prescriptions in second/third trimester or in the 30 days before labor were

associated with an increased risk for obesity in the offspring at school age [13,14]. Similarly, increased risk

for obesity in conscripts of mothers with infections during pregnancy was reported in a Danish register-based

study [15]. Continuing after birth, six recent observational studies found increased prevalence of obesity or

increased BMI following antibiotics treatment of otherwise healthy children from western countries [16–21].

In a case-control study from Canada, exposure to antibiotics before 12 months of age increased the risk of

overweight and central obesity in children at age nine and 12 years more than two-fold [20]. Exposure to

more than three antibiotics courses in the first two years of life increased the risk of obesity at age three in

US children [18] and exposure to antibiotics before six months of age increased both height and bodyweight

in Finnish children [21].

Interestingly, subgroup and interaction analyses in these studies also raised the possibility of drug, host or

maternal factors modifying the effect of antibiotics on bodyweight: children of mothers with high maternal

pre-pregnancy BMI had a decreased risk of overweight with exposure to antibiotics in infancy, whereas

children of normal-weight mothers exposed to antibiotics early in life had an increased risk of later

This article is protected by copyright. All rights reserved.

 overweight in a Danish birth cohort study. Associations between obesity and exposure to broad-spectrum but

not narrow-spectrum antibiotics were reported in another study, suggesting the obesity risk to be dependent

on the antimicrobial spectrum [18]. Finally, four studies found notable, yet unexplained gender differences
Accepted Article
with stronger associations between early life antibiotics and overweight/obesity among boys compared to

girls [17,19–21].

Although these studies in children are worrying, it should be emphasized that the observational design used,

does not allow inference of causality: Exposure to antibiotics was not only associated with development of

obesity, but also strongly associated with a long list of more or less unexpected factors such as parity,

socioeconomic status, maternal BMI, duration of breastfeeding, maternal smoking status, presence of

maternal asthma etc. [16,17,20,21]. Although vigorous adjustments for differences in these known factors

were performed to reduce risk of confounding [16,17,20,21], it is impossible to rule out, that children, who

developed obesity, were already at increased risk for developing infections in early life due to differences in

factors not adjusted for in the statistical models. At the same time, associations between adult BMI and

overall infections [15,22] as well as viral infections [23], have been reported in other studies, raising the

possibility that it may be the inflammatory response or other non-antibiotic-related factors, that mediate this

association to overweight or obesity [24].

Finally, in three of the largest observational studies [17,18,25], exposure to antibiotics before 6 months of

age did not significantly affect the risk of obesity at age seven following adjustment for potential

confounders [17,25], and exposure to narrow-spectrum antibiotics or exposure to less than four courses of

antibiotics before two years of age did not affect the risk of obesity [18].

Studies in adults

In adults, the effect of antibiotics on bodyweight profile has been examined in three observational studies

and three controlled prospective trials.

This article is protected by copyright. All rights reserved.

 In a small case-control study of individuals admitted to a French hospital with suspected endocarditis, cases

that had the diagnosis confirmed and received antibiotics were more prone to develop weight gain one year

later than control subjects in whom the diagnosis was rejected and no antibiotics was used [26]. A subgroup
Accepted Article
analysis suggested that the combination of vancomycin and gentamycin in particular was associated with

weight gain [26]. However, baseline characteristics (including BMI) in the case and control groups were

quiet different, raising the possibility for selection bias as explanation for the findings [26]. A similar

observational study by the same research group assessed changes in bodyweight in 48 cases with Q-fever

endocarditis before and after long-term treatment with doxycycline and hydroxychloroquine, in comparison

to 34 control participants not treated with antibiotics (outpatients referred to the infectious disease unit) [27].

After one year of follow-up, weight gain was significantly more common in the antibiotics-treated group, but

there was no difference in body weight between the two populations since weight loss was also more

common among the treated patients [27].

In a prospective cohort study of veterans referred to elective upper endoscopy, a significant weight gain and

an increase in the plasma levels of ghrelin was seen in the group who received antibiotic therapy for H.

pylori eradication (14-day twice-daily regimen of amoxicillin 1,000 mg, clarithromycin 500 mg), compared

with no weight gain in a group of matched control subjects not treated with antibiotics [28]. In line, when a

large group of unselected patients with H. pylori (n=1558) where randomized to standard eradication therapy

or placebo, there was an increased weight gain in the intervention group compared to the control. However,

there was also a reduced prevalence of dyspepsia in the group eradicated for H. pylori, which could have

confounded the results upon causing increased food intake [29].

In addition to these four studies examining effects of antibiotics in participants with documented pathogen

colonization or infection, two interventional studies have assessed the effect of antibiotics in otherwise

healthy adults. In a large randomized controlled trial conducted in 1955, navy recruits (n=330) without

apparent infections and a mean BMI of 22.3 kg/m2 were randomized to receive either penicillin, tetracycline

or placebo once daily for seven weeks [30]. An increased weight gain was observed for the two groups

treated with antibiotics compared to the group receiving placebo following seven weeks. Unfortunately, the

This article is protected by copyright. All rights reserved.

 study was not initially designed to evaluate effects on bodyweight; there were no reports on prevalence of

obesity or adiposity, and height and bodyweight was not measured by a predefined study protocol [30]. Also,

a second sub-study by the same authors failed to reproduce the initial findings on bodyweight [30].
Accepted Article
In a recent interventional study, we tested the metabolic impact of a broad-spectrum antibiotic cocktail

assumed to suppress the general intestinal bacterial load. Twelve lean and healthy young males were

subjected to four days of treatment with the combination of orally administered vancomycin (500 mg),

gentamycin (40 mg) and meropenem (500 mg) given once-daily. Suppression in gut bacteria abundance was

confirmed by cultivation of stool samples. In this study mean bodyweight of the participants slightly

increased from 78.1 kg (95% Confidence Intervals (CI) 73.9-82.4 kg) at baseline to 79.4 kg (95% CI 78.2-

80.6 kg) 180 days following the antibiotics exposure. Since there was no placebo-treated group for

comparison in this study, it is impossible to rule out random variation or a general tendency towards weight

gain over time as explanation for the observed weight gain. No changes in resting energy expenditure or

amount of food ingested during ad libitum meal tests were seen [31].

Taken together, a number of observational and non-randomized clinical studies support the possibility that

antibiotics exposure, in particular early life exposure, may lead to increases in BMI or prevalence of obesity.

Nevertheless, it is important to emphasize that neither of these studies provided causal or mechanistic

evidence for the suggested effects of antibiotics on bodyweight.

Effects of antibiotics on glucose metabolism

Although there have been previous reports on risk of hypoglycemia as well as hyperglycemia with antibiotic

treatments, these studies mostly have focused on pharmacodynamic interactions between glucose-lowering

agents and antimicrobials in patients diagnosed with diabetes [32,33]. Until recently, the association between

human glucose metabolism and antibiotic treatment was unexplored.

This article is protected by copyright. All rights reserved.

 Observational studies

Two large population-based case-control studies recently suggested links between exposure to antibiotics

and subsequent development of diabetes. One of the studies combined data from a general practitioner-based
Accepted Article
database and identified 208,002 cases with new onset diabetes (of any kind) and 815,576 matched control

subjects free of diabetes from a UK population [34]. After multifactor-adjustment for potential confounders,

there was an increased risk for diabetes with increasing exposure to all of the examined groups of antibiotics,

but an isolated estimate for the risk of type 2 diabetes was not provided and the concept of a combined main

analysis for type 1 diabetes and type 2 diabetes (and other forms of diabetes) could be questioned by the

quite marked differences in etiology and pathogenesis of the different forms of diabetes.

In a population-based Danish case-control study, 179,504 cases with new onset type 2 diabetes were

identified and age and gender-matched with 1,364,008 control subjects free of diabetes using the Danish

Prescription Registry, Danish Patient Registry and Statistics of Denmark [35]. In this study, odds ratio for

type 2 diabetes was 1.53 with redemption of ≥5 vs 0-1 redemptions of antibiotics of any type. A moderate

and rather homogenous dose-response was seen for all types and commonly used groups of antibiotics, but

the association was slightly stronger with narrow-spectrum and bactericidal antibiotics (odds ratio 1.55 and

1.48) compared to broad-spectrum and bacteriostatic types of antibiotics (odds ratio 1.31 and 1.39). The

overexposure to antibiotics in the case group was seen up to 15 years before diagnosis reducing the

possibility for an increased antibiotics demand due to prediabetes or undiagnosed type 2 diabetes. Still, as in

the above-mentioned observational studies in children, the possibility exists that cases were at an increased

risk of developing infections years before type 2 diabetes diagnosis. In particular, it could be speculated

whether cases had more frequent obesity or overweight already 15 years before their type 2 diagnosis, and as

a consequence of this, showed increased risk of infection and, thus antibiotics use, already at this point [36].

This article is protected by copyright. All rights reserved.

 Interventional studies

Only three human interventional studies have examined the association between antibiotics and glucose

metabolism. In a randomized, double-blinded study, 20 obese males with the metabolic syndrome were
Accepted Article
randomized to receive either vancomycin or amoxicillin for seven days (both drugs 500 mg thrice-daily)

[37]. Hyperinsulinemic euglycemic clamps and oral glucose tolerance tests were performed before and

immediately after the antibiotics course. No changes in glucose tolerance were observed with any of the

antibiotics, but a reduction in peripheral insulin sensitivity was found following vancomycin but not

amoxicillin treatment. No significant changes were seen in body weight, plasma lipids or the gut hormones

glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). In an attempt to

replicate these findings, the effect of placebo, vancomycin or amoxicillin for seven days (in the same

dosages as above), were investigated in 50 patients with obesity and prediabetes. Again, hyperinsulinemic,

euglycaemic clamps with glucose tracer infusions were performed before and immediately after the

antibiotics treatment, but this time, no significant changes were observed in insulin sensitivity or insulin-

stimulated hepatic glucose disposal in any of the three study arms [38]. Of note, none of these two studies

have provided data on long-term follow-up.

In our aforementioned interventional study, where a cocktail of vancomycin, gentamycin and meropenem

where given for four days, no short or long-term changes were seen in glucose tolerance, and apart from a

reversible rise in the fasting and postprandial levels of peptide YY (PYY), no changes in gut or pancreatic

hormones were found [31].

In summary, there is observational evidence for an association between antibiotics exposure in adulthood

and subsequent development of type 2 diabetes, whereas the three controlled interventional studies on adults

did not find any significant impact of antibiotics on fasting plasma glucose levels, endogenous glucose

production, postprandial glucose tolerance or hepatic insulin sensitivity.

Table 1 and 2 give an overview of the existing observational and interventional human studies examining the

effects of antibiotics on bodyweight regulation, glucose metabolism and development of obesity or diabetes.

This article is protected by copyright. All rights reserved.

 As previously noted, a number of interventional studies have demonstrated changes in bodyweight following

antibiotic treatment of children with disposition to infection or concomitant infections; these studies are

reviewed elsewhere [12].

Accepted Article

Effects of antibiotics on the human gut microbiota

Assessment of the effects of antibiotics on gut microbiota

The ability of antibiotics to eradicate gut bacteria was documented shortly after the discovery of penicillin

[39]. Pioneering cultivation-based studies characterized the antibiotic-induced changes in dominant groups

of gut bacteria and showed that the eviction of microorganisms sensitive to the antibiotics used could provide

space for resistant strains or foreign microorganisms to occupy and dominate niches previously occupied by

commensal microbes, reviewed in [40]. Although cultivation-based techniques for gut microbiota

assessments are still improving [41], it is difficult and time-consuming to cultivate the vast amounts of

bacteria in the gut. With the technological development and reduced price for next-generation sequencing,

targeted or untargeted sequencing of bacterial DNA is now widely used to assess the composition and

function of the gut microbiota [42].

Effects of antibiotics on gut microbial ecology

In recent studies using molecular techniques based on analysis of the prokaryotic 16S ribosomal RNA (16S

rRNA) gene, loss of diversity and dramatic short-term changes in several bacterial taxa have been a

consistent finding following antibiotics exposure [37,43–48]. Long-term effects on microbial composition

have also been reported in some studies, but with marked inter-individual differences and even intra-

individual differences in the response to the same antibiotic [45]. Thus, substantial relative changes in

microbial abundances were seen several months after cessation of antibiotics treatment in some studies [43–

45,48,49], whereas other studies demonstrated more modest effects on absolute bacterial numbers following

This article is protected by copyright. All rights reserved.

 antibiotics treatment [37,50]. Reductions in the total number of gut bacteria seem expected following

antibiotic treatment, but, intriguingly, studies using quantitative PCR of the 16S rRNA gene to establish the

total number of bacteria have reported unchanged [37,50] or even a tendency towards increased number of
Accepted Article
bacteria [46] following broad-spectrum antibiotics treatment.

Many of the studies published so far have investigated only few subjects and are characterized by significant

heterogeneity with respect to subject characteristics (including differences in ethnicity and age), type of

antibiotic(s) tested, methods applied to study the compositional changes, and length of follow-up time. Route

of antibiotic administration (intravenous vs. oral) and age at the time of exposure seem to influence the

effects of antibiotics on the microbiome [51,52]. Variation resulting from changes in diet, lifestyle and/or use

of other prescribed medications over time may have a similarly strong effect on the microbiota [53–55] and

together the changes in such covariates and the disparities in the studies mentioned above complicate

prediction of an individual’s microbial response to a particular antibiotics course. In addition, it is possible

that the antibiotics-induced effects on gut microbial ecology cannot be elucidated with the application of 16S

rRNA gene sequencing techniques alone. As an example, the non-absorbable antibiotic rifaximin used to

treat hepatic encephalopathy in patients with liver cirrhosis seems to confer marked changes in gut

microbiota function (changes in the serum metabolome) with no or only very limited impact on the

microbiota composition as assessed by 16S rRNA gene sequencing [56]. Two recent studies have used

metagenomics prediction tools or untargeted sequencing of the collective bacterial genome (the microbiome)

to elucidate the effects of antibiotics on the human gut microbiota [48,57]. In one of these studies, 18 healthy

adults were submitted to a seven-day course of a second-generation cephalosporin, with collection of fecal

samples for DNA extraction before, immediately after and 90 days after the antibiotic course [57]. A

decrease in the abundance of several bacterial families was seen immediately after the antibiotic course, but

the microbiome generally recovered to its initial state after 90 days. Notably, the inter-individual variability

at the species level (the lowest level in the taxonomic hierarchy) was greater than the effect of the antibiotic

for most participants.. In the other study, 74 healthy volunteers were randomized to receive clindamycin,

ciprofloxacin, amoxicillin, minocycline or placebo for 5-10 days [48]. Changes in the microbiota

This article is protected by copyright. All rights reserved.

 composition where seen immediately following all four types of antibiotics, but in contrast to amoxicillin

and minocycline, clindamycin and ciprofloxacin resulted in pronounced and significant changes in bacterial

composition, as well as reduction in bacterial diversity for up till four and 12 months following exposure,
Accepted Article
respectively. Some degree of functional redundancy (different groups of bacteria perform the same function)

was observed, but metagenomics predictions also suggested sustained functional changes following

clindamycin and ciprofloxacin exposure, with notable reductions in fermentative butyrate-producing

pathways [48].

Pathways through which the gut microbiota may affect host metabolism

The pathways through which the gut microbiota may affect metabolic regulation and energy balance of its

host are only partially understood. Most of the evidence is derived from rodent models, where a complete

microbiota modulation is possible, either by germ-free rearing, treatment with long-term and broad-spectrum

antibiotics, dietary interventions or inoculation with specific gut microbes from other rodents or even

humans.

From basic thermodynamic considerations, antibiotic treatment may only affect energy balance if it alters

energy intake or energy expenditure. We are aware of one only human trial measuring energy intake or

energy expenditure (though not with golden standard techniques) before and after antibiotics treatment, and

in this study no differences were seen [31]. As a more fundamental limitation to the application of such

simple energy equations, there is evidence from animal models, that weight gain and increased lipid

deposition may result from increased microbial capacity for energy harvesting or energy deposition even

with decreased energy intake and increased expenditure [5,6]. In line with this, mice that received long-term

antibiotic treatment developed increased adiposity, but decreased caloric output in fecal samples, compared

with untreated mice eating the same amounts of calories [58]. Similarly, treatment of mice with high dosages

of broad-spectrum antibiotics induced an increase in small intestinal length and absorptive area as a

This article is protected by copyright. All rights reserved.

 mechanism assumed to increase host absorptive capacity and compensate for the loss of energy harvesting

potential following eradication of gut bacteria [59].

In the following we will discuss just three microbiota-affected metabolic pathways, selected as they are well-
Accepted Article
validated in human (as well as animal) experiments and shown to be susceptible to antibiotics treatment. A

short summary of these pathways is depicted in Figure 1, but it should be noted, that the gut microbiota has

been implicated in numerous other host physiologic and metabolic processes that do not fall within the scope

of this review.

Short-chain fatty acids

Unlike the human genome, the gut microbiome encodes many enzymes necessary for degrading complex

plant polysaccharides in the diet [60]. Gut bacteria in the colon ferment these to short-chain fatty acids,

which represent an important energy source and have been estimated to cover as much as 10% of daily

energy requirements in humans on a typical western diet [61,62]. Rodents raised under germ-free conditions

without intestinal bacteria, have reduced levels of intestinal short-chain fatty acids, increased mortality and

decreased generation of ketone bodies from short-chain fatty acids during starvation [63]. In addition to this

direct caloric contribution, short-chain fatty acids have an equally important effect on metabolic and energy

balances by altering expression and secretion of gut hormones involved in multiple aspects of postprandial

physiology, intestinal barrier function and gut-brain crosstalk influencing appetite and food intake [64–68].

In animal models, changes in the availability of intestinal short-chain fatty acids has been shown to affect

blood levels of the gut hormones GLP-1, glucagon-like peptide-2 (GLP-2) and peptide YY (PYY) with

ensuing effects on food intake, weight gain, insulin signaling, glucose tolerance, intestinal barrier function

and intestinal transit time [64,65,67–71]. When antibiotics are given to modulate the gut microbiota in

rodents, altered production of short-chain fatty acids [58,59,71–73] and marked changes in gut hormone

levels are seen [58,71].

In humans, ingestion of prebiotics (complex carbohydrates stimulating the growth or activity of gut microbes

[74]), which are fermented to short-chain fatty acids by the gut microbiota, has been shown to increase

This article is protected by copyright. All rights reserved.

 plasma levels of GLP-1, GLP-2 and PYY [75–77], while inhibiting release of the appetite-stimulating

hormone ghrelin [75,76] and increasing subjective satiety feeling [78]. In contrast, short-term treatment with

broad-spectrum antibiotics, reduces the fecal levels of short-chain fatty acids [79,80] and reduces butyrate-
Accepted Article
producing pathways in the gut microbiome of healthy volunteers [48].

In some cross-sectional studies, inverse associations between abundance of butyrate-producing bacteria and

prevalence of type 2 diabetes or insulin resistance have been reported [2,3]. However, the metabolic outcome

of generating short-chain fatty acids remains elusive since fecal excretion of short-chain fatty acids have

been shown to be increased in patients with obesity [81,82] and following a high-fat diet [83], but decreased

along with increasing insulin sensitivity in obese males receiving a transfer of fecal material from lean

glucose-tolerant individuals [7].

Bile acids

It has become apparent that bile acids are involved in a number of activities beyond their classic role in lipid

digestion and absorption. Bile acids have been identified as ligands of the nuclear farnesoid X receptor

(FXR) and the membrane-bound, G protein-coupled bile acid receptor 1 (TGR5), both of which seem to play

important roles in the regulation of gut hormone secretion and glucose and lipid metabolism [84–86]. The

potency of bile acids to stimulate these receptors, however, is highly variable between different bile acids,

with a tendency towards higher agonist potency of the secondary bile acids [37,86]. In that respect it is

interesting that bacterial biotransformations of bile acids, including deconjugation of bile acids and

dehydroxylation of primary to secondary bile acids, are essential for the composition of bile acids in the

enterohepatic circulation [87]. Germ-free rodents and conventionally raised rodents treated with broad-

spectrum antibiotics display marked alterations in global bile acid metabolism: Compared with

conventionally raised or untreated rodents they have an increased bile acid pool, reduced amounts of

secondary bile acids, increased biliary secretion of bile acids, increased intestinal bile acid reabsorption and

decreased fecal excretion of bile acids [88–90]. Moreover, altered FXR and TGR5 signaling have been

shown following antibiotic-induced gut microbiota disturbances [89]. In humans, antibiotic treatment has

This article is protected by copyright. All rights reserved.

 been shown to alter fecal and serum levels of bile acids [37,91,92]. Furthermore, antibiotics have

documented effects for the treatment of abnormal secondary bile acids levels in patients with cholesterol

super-saturated gall stones [91,92].

Accepted Article
At the same time that gut bacteria modulate the bile acid pool, bile acids also signal back on the gut

microbiota. As a result of detergent effects, bile acids have antimicrobial and cell membrane destabilizing

effects in the gut at physiological concentrations [93]. In rodents, increased intestinal bile acid levels have

been shown to lead to marked simplifications of the gut microbiota composition with selection or

overgrowth of microorganisms that are bile acid tolerant at the expense of more bile acid sensitive

commensal bacteria [94,95]. Accordingly, when intestinal excretion of bile acid is prevented by bile duct

ligation in animal models, bacterial overgrowth in the small intestine is seen [96]. Likewise, the reduced

capacity for hepatic bile acid excretion in liver cirrhosis has been proposed to explain some of the intestinal

bacterial overgrowth seen in rodent models [97] and humans with liver cirrhosis [98–100].

An exciting perspective of these observations is that bile acids can be seen as mediators of the host-microbe

crosstalk, capable of influencing both host physiologic pathways and microbial community [101]:

Environmental changes such as altered dietary intake can drive changes in host immune response [95] and

host metabolic homeostasis [101] through a bile acid-mediated change in gut microbiota composition.

Likewise, hepatic or metabolic diseases may change the biliary excretion with consequent impact on the

microbiota.

Low-grade inflammation

Previously, it was believed that healthy individuals without clinical signs of infection were sterile, or free of

microorganisms within their blood and extraintestinal organs, but with the recent advances in bacterial

identification, live bacteria or bacterial components have been observed in blood, visceral adipose tissue and

even atherosclerotic plaques of “clinically uninfected” individuals [102–105].

This article is protected by copyright. All rights reserved.

 Translocation of bacteria or bacterial components through the intestinal barrier leads to activation of cells in

the innate immune system and consequent initiation of an inflammatory cascade. Insulin resistance is a well-

established consequence of severe inflammation as observed in sepsis [106], but low-grade inflammation
Accepted Article
also induces insulin resistance and glucose intolerance even without infection: Mice chronically infused with

a low dose of lipopolysaccharide, a component from the outer membrane of gram-negative bacteria,

developed systemic and adipose tissue inflammation and impaired glucose tolerance together with increased

body weight and hepatic lipid deposition [107,108]. Similarly, glucose-tolerant adults displayed adipose

tissue inflammation and insulin resistance up to 24 hours after infusion with lipopolysaccharide [109].

In a large cohort of individuals free of diabetes at study entry, the presence of bacterial DNA in blood was

shown to be an independent risk factor for development of type 2 diabetes [103]. Also, concentration of

bacterial DNA in blood and concentration of lipopolysaccharide-binding protein in adipose tissue correlated

positively with insulin resistance and adipose tissue inflammation but negatively with weight loss in morbid

obese patients undergoing bariatric surgery [104,110].

Interestingly, broad-spectrum antibiotics salvaged mice from diet-induced low-grade inflammation and

insulin resistance, presumably upon eradication of some of the gut pathobionts induced by high-fat feeding

[72,111]. In humans, the non-absorbable antibiotic rifaximin has been successfully applied to reduce

inflammation and decrease the absorption of gut endotoxins in patients with chronic liver cirrhosis [56,98].

Together, these results support the perspective that antibiotics-induced gut microbiota modulations may also

lead to metabolic improvements, and perhaps in particular if the existing gut microbiota is disturbed.

Concluding remarks

The gut microbiota has been identified as a substantial and causal driver of metabolic phenotype in animal

models, in part by controlling the production of short-chain fatty acids and influencing the composition of

This article is protected by copyright. All rights reserved.

 bile acids and the inflammatory level of the host. Antibiotics affect the composition and the function of

human gut microbiota. There is mounting observational evidence for an association between antibiotics

exposure in infancy and development of obesity in childhood, but it has not been studied in randomized
Accepted Article
controlled trials whether there could be long-term metabolic effect of antibiotics when given to children free

of infections or chronic diseases. There is also observational evidence for an association between antibiotics

exposure in adulthood and subsequent development of type 2 diabetes. However, in three controlled

interventional studies on adults with or without impaired metabolic regulation, antibiotics had no effect on

fasting plasma glucose levels, endogenous glucose production, postprandial glucose tolerance or hepatic

insulin sensitivity. Thus, based on the existing literature, it seems plausible that antibiotics treatment can

influence metabolic homeostasis in humans, but there is insufficient evidence to claim that individual

antibiotics or antibiotics in general may confer clinically significant short-term effects on glucose

metabolism or bodyweight control if given to otherwise healthy and uninfected adults. In the light of the

widespread use of antibiotics and the increasing global burden of metabolic diseases, we believe that a

randomized placebo-controlled clinical trial of the short- and long-term effects of a commonly used

antibiotic is ethically justified in adult humans free of infection. Such a study should clarify whether

antibiotics have clinically relevant effects on energy balance or glucose metabolism. At the same time,

carefully designed cohort studies of patients who are inevitably receiving antibiotics should also be initiated

in order to improve our understanding of the mechanisms through which antibiotics-induced changes in the

gut microbiota may affect human metabolism and bodyweight, and such studies should specifically be used

to improve our understanding of the effect of early life antibiotic exposure.

This article is protected by copyright. All rights reserved.

 References

1. Xu J, Gordon JI. Honor thy symbionts. Proc Natl Acad Sci 2003; 100: 10452–10459.

2. Qin J, Li Y, Cai Z, et al. A metagenome-wide association study of gut microbiota in type 2

Accepted Article
diabetes. Nature 2012; 490: 55–60.

3. Karlsson FH, Tremaroli V, Nookaew I, et al. Gut metagenome in European women with
normal, impaired and diabetic glucose control. Nature 2013; 498: 99–103.

4. Le Chatelier E, Nielsen T, Qin J, et al. Richness of human gut microbiome correlates with
metabolic markers. Nature 2013; 500: 541–546.

5. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-
associated gut microbiome with increased capacity for energy harvest. Nature 2006; 444:
1027–1031.

6. Bäckhed F, Ding H, Wang T, et al. The gut microbiota as an environmental factor that
regulates fat storage. Proc Natl Acad Sci U S A 2004; 101: 15718–15723.

7. Vrieze A, Van Nood E, Holleman F, et al. Transfer of intestinal microbiota from lean donors
increases insulin sensitivity in individuals with metabolic syndrome. Gastroenterology; 143:
913–916.e7.

8. Ridaura VK, Faith JJ, Rey FE, et al. Gut Microbiota from Twins Discordant for Obesity
Modulate Metabolism in Mice. Science 2013; 341: 1241214–1241214.

9. Gustav Martin. p-Aminobenzoic Acid and Sulfonamides in Rat Nutrition. Exp Biol Med
1942: 56–59.

10. Dibner JJ, Richards JD. Antibiotic growth promoters in agriculture: history and mode of
action. Poult Sci 2005; 84: 634–643.

11. Saiman L, Anstead M, Mayer-Hamblett N, et al. Effect of azithromycin on pulmonary

function in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa: a
randomized controlled trial. Jama 2010; 303: 1707–1715.

12. Gough EK, Moodie EEM, Prendergast AJ, et al. The impact of antibiotics on growth in
children in low and middle income countries: systematic review and meta-analysis of
randomised controlled trials. BMJ 2014; 348: g2267–g2267.

13. Mueller NT, Whyatt R, Hoepner L, et al. Prenatal exposure to antibiotics, cesarean section
and risk of childhood obesity. Int J Obes 2015; 39: 665–670.

14. Mor A, Antonsen S, Kahlert J, et al. Prenatal exposure to systemic antibacterials and
overweight and obesity in Danish schoolchildren: a prevalence study. Int J Obes 2015; 39:
1450-55.

This article is protected by copyright. All rights reserved.

 15. Cocoros NM, Lash TL, Nørgaard M, Farkas DK, DeMaria A, Sørensen HT. Hospitalized
prenatal and childhood infections and obesity in Danish male conscripts. Ann Epidemiol
2013; 23: 307–313.

16. Trasande L, Blustein J, Liu M, Corwin E, Cox LM, Blaser MJ. Infant antibiotic exposures
and early-life body mass. Int J Obes 2013; 37: 16–23.
Accepted Article
17. Ajslev TA, Andersen CS, Gamborg M, Sørensen TIA, Jess T. Childhood overweight after
establishment of the gut microbiota: the role of delivery mode, pre-pregnancy weight and
early administration of antibiotics. Int J Obes 2011; 35: 522–529.

18. Bailey LC, Forrest CB, Zhang P, Richards TM, Livshits A, DeRusso PA. Association of
Antibiotics in Infancy With Early Childhood Obesity. JAMA Pediatr 2014; 168: 1063-69..

19. Murphy R, Stewart AW, Braithwaite I, Beasley R, Hancox RJ, Mitchell EA. Antibiotic
treatment during infancy and increased body mass index in boys: an international cross-
sectional study. Int J Obes 2014; 38: 1115–1119.

20. Azad MB, Bridgman SL, Becker AB, Kozyrskyj AL. Infant antibiotic exposure and the
development of childhood overweight and central adiposity. Int J Obes 2014; 38: 1290–1298.

21. Saari A, Virta LJ, Sankilampi U, Dunkel L, Saxen H. Antibiotic Exposure in Infancy and
Risk of Being Overweight in the First 24 Months of Life. Pediatrics 2015; 135: 617–626.

22. Burgner D, Sabin M, Magnussen, C, et al. Infection-related hospitalization in childhood and

adult metabolic outcomes. Pediatrics 2015; 136. 554-62.

23. Yamada T, Hara K, Kadowaki T. Association of Adenovirus 36 Infection with Obesity and
Metabolic Markers in Humans: A Meta-Analysis of Observational Studies. Targher G, editor.
PLoS ONE 2012; 7: e42031.

24. Lassenius MI, Pietilainen KH, Kaartinen K, et al. Bacterial Endotoxin Activity in Human
Serum Is Associated With Dyslipidemia, Insulin Resistance, Obesity, and Chronic
Inflammation. Diabetes Care 2011; 34: 1809–1815.

25. Blustein J, Attina T, Liu M, et al. Association of caesarean delivery with child adiposity from
age 6 weeks to 15 years. Int J Obes 2013; 37: 900–906.

26. Thuny F, Richet H, Casalta J-P, Angelakis E, Habib G, Raoult D. Vancomycin Treatment of
Infective Endocarditis Is Linked with Recently Acquired Obesity. Bereswill S, editor. PLoS
ONE 2010; 5: e9074.

27. Angelakis E, Million M, Kankoe S, et al. Abnormal Weight Gain and Gut Microbiota
Modifications Are Side Effects of Long-Term Doxycycline and Hydroxychloroquine
Treatment. Antimicrob Agents Chemother 2014; 58: 3342–3347.

28. Francois F, Roper J, Joseph N, et al. The effect of H. pylori eradication on meal-associated
changes in plasma ghrelin and leptin. BMC Gastroenterol 2011; 11: 37.

This article is protected by copyright. All rights reserved.

 29. Lane JA, Murray LJ, Harvey IM, Donovan JL, Nair P, Harvey RF. Randomised clinical trial:
Helicobacter pylori eradication is associated with a significantly increased body mass index
in a placebo-controlled study. Aliment Pharmacol Ther 2011; 33: 922–929.

30. Haight TH, Pierce WE. Effect of Prolonged Antibiotic Administration on the Weight of
Healthy Young Males One Figure. J Nutr 1955; 56: 151–161.
Accepted Article
31. Mikkelsen KH, Frost M, Bahl MI, et al. Effect of Antibiotics on Gut Microbiota, Gut
Hormones and Glucose Metabolism. PLOS ONE 2015; 10: e0142352.

32. Chou H-W, Wang J-L, Chang C-H, Lee J-J, Shau W-Y, Lai M-S. Risk of Severe
Dysglycemia Among Diabetic Patients Receiving Levofloxacin, Ciprofloxacin, or
Moxifloxacin in Taiwan. Clin Infect Dis 2013; 57: 971–980.

33. Mohr JF, McKinnon PS, Peymann PJ, Kenton I, Septimus E, Okhuysen PC. A Retrospective,
Comparative Evaluation of Dysglycemias in Hospitalized Patients Receiving Gatifloxacin,
Levofloxacin, Ciprofloxacin, or Ceftriaxone. Pharmacother J Hum Pharmacol Drug Ther
2005; 25: 1303–1309.

34. Boursi B, Mamtani R, Haynes K, Yang Y-X. The effect of past antibiotic exposure on
diabetes risk. Eur J Endocrinol 2015; 172: 639–648.

35. Mikkelsen KH, Knop FK, Frost M, Hallas J, Pottegård A. Use of Antibiotics and Risk of
Type 2 Diabetes: A Population-Based Case-Control Study. J Clin Endocrinol Metab 2015;
100: 3633-40.

36. Falagas ME, Kompoti M. Obesity and infection. Lancet Infect Dis 2006; 6: 438–446.

37. Vrieze A, Out C, Fuentes S, et al. Impact of oral vancomycin on gut microbiota, bile acid
metabolism, and insulin sensitivity. J Hepatol 2014; 60: 824–831.

38. Reijnders D, Goosens G, Neis E, et al. Effects of gut microbiota manipulation by antibiotics
on host metabolism in obese humans. Conference abstract, EMBL, Heidelberg, Germany;
2015.

39. Di Caprio JM, Rantz LA. Effects of terramycin on the bacterial flora of the bowel in man.
AMA Arch Intern Med 1950; 86: 649–657.

40. Nord CE. The effect of antimicrobial agents on the ecology of the human intestinal
microflora. Vet Microbiol 1993; 35: 193–197.

41. Rettedal EA, Gumpert H, Sommer MOA. Cultivation-based multiplex phenotyping of human
gut microbiota allows targeted recovery of previously uncultured bacteria. Nat Commun
2014; 5: 4714.

42. Kuczynski J, Lauber CL, Walters WA, et al. Experimental and analytical tools for studying
the human microbiome. Nat Rev Genet 2011; 13: 47–58.

43. Jernberg C, Löfmark S, Edlund C, Jansson JK. Long-term ecological impacts of antibiotic
administration on the human intestinal microbiota. ISME J 2007; 1: 56–66.

This article is protected by copyright. All rights reserved.

 44. Jakobsson HE, Jernberg C, Andersson AF, Sjölund-Karlsson M, Jansson JK, Engstrand L.
Short-Term Antibiotic Treatment Has Differing Long-Term Impacts on the Human Throat
and Gut Microbiome. PLoS ONE 2010; 5: e9836.

45. Dethlefsen L, Relman DA. Colloquium Paper: Incomplete recovery and individualized
responses of the human distal gut microbiota to repeated antibiotic perturbation. Proc Natl
Accepted Article
Acad Sci 2010; 108: 4554–4561.

46. Panda S, El khader I, Casellas F, et al. Short-Term Effect of Antibiotics on Human Gut
Microbiota. PLoS ONE 2014; 9: e95476.

47. Pallav K, Dowd SE, Villafuerte J, et al. Effects of polysaccharopeptide from \iTrametes
Versicolor and amoxicillin on the gut microbiome of healthy volunteers: A randomized
clinical trial. Gut Microbes 2014l; 5: 458–467.

48. Zaura E, Brandt BW, Teixeira de Mattos MJ, et al. Same Exposure but Two Radically
Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus Long-
Term Microbial Shifts in Feces. mBio 2015; 6: e01693–15 – e01693–15.

49. Cochetière MFDL, Durand T, Lepage P, Bourreille A, Galmiche JP, Doré J. Resilience of the
Dominant Human Fecal Microbiota upon Short-Course Antibiotic Challenge. J Clin
Microbiol 2005; 43: 5588–5592.

50. Fouhy F, Guinane CM, Hussey S, et al. High-Throughput Sequencing Reveals the
Incomplete, Short-Term Recovery of Infant Gut Microbiota following Parenteral Antibiotic
Treatment with Ampicillin and Gentamicin. Antimicrob Agents Chemother 2012; 56: 5811–
5820.

53. Arat S, Spivak A, Horn SV, et al. Microbiome Changes in Healthy Volunteers Treated with
GSK1322322, a Novel Antibiotic Targeting Bacterial Peptide Deformylase. Antimicrob
Agents Chemother 2015; 59: 1182–1192.

59. Arboleya S, Sánchez B, Milani C, et al. Intestinal Microbiota Development in Preterm

Neonates and Effect of Perinatal Antibiotics. J Pediatr 2015; 166: 538–544.

53. Clarke SF, Murphy EF, O’Sullivan O, et al. Exercise and associated dietary extremes impact
on gut microbial diversity. Gut 2014: 63; 1913-20.

54. Forslund K, Hildebrand F, Nielsen T, et al. Disentangling type 2 diabetes and metformin
treatment signatures in the human gut microbiota. Nature 2015; 528: 262–266.

55. Wu GD, Chen J, Hoffmann C, et al. Linking long-term dietary patterns with gut microbial
enterotypes. Science 2011; 334: 105–108.

51. Bajaj JS, Heuman DM, Sanyal AJ, et al. Modulation of the Metabiome by Rifaximin in
Patients with Cirrhosis and Minimal Hepatic Encephalopathy. PLoS ONE 2013; 8; 60042.

52. Raymond F, Ouameur AA, Déraspe M, et al. The initial state of the human gut microbiome
determines its reshaping by antibiotics. ISME J 2015; doi: 10.1038.

This article is protected by copyright. All rights reserved.

 71. Cho I, Yamanishi S, Cox L, et al. Antibiotics in early life alter the murine colonic
microbiome and adiposity. Nature 2012; 488: 621–626.

59. Chevalier C, Stojanović O, Colin DJ, et al. Gut Microbiota Orchestrates Energy Homeostasis
during Cold. Cell 2015 Dec 3; 163: 1360–1374.
Accepted Article
60. Gill SR, Pop M, DeBoy RT, et al. Metagenomic Analysis of the Human Distal Gut
Microbiome. Science 2006; 312: 1355–1359.

61. Cummings JH, Pomare EW, Branch WJ, Naylor CP, Macfarlane GT. Short chain fatty acids
in human large intestine, portal, hepatic and venous blood. Gut 1987; 28: 1221–1227.

62. \bMacfarlane G, Gibson G. \iCarbohydrate fermentation, energy transduction and gas

metabolism in the human large intestine. In: Mackie RI, White BA (eds) Gastrointestinal
Microbiology, vol 1. Chapman and Hall, pp 269-318. Springer; 1997.

63. Crawford PA, Crowley JR, Sambandam N, et al. Regulation of myocardial ketone body
metabolism by the gut microbiota during nutrient deprivation. Proc Natl Acad Sci U S A
2009; 106: 11276–11281.

64. Lin HV, Frassetto A, Kowalik Jr EJ, et al. Butyrate and Propionate Protect against Diet-
Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent
Mechanisms. PLoS ONE 2012: e35240.

65. Tolhurst G, Heffron H, Lam YS, et al. Short-Chain Fatty Acids Stimulate Glucagon-Like
Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2. Diabetes 2011; 61: 364–
371.

66. Duca FA, Lam TKT. Gut microbiota, nutrient sensing and energy balance. Diabetes Obes
Metab 2014; 16: 68–76.

67. Samuel BS, Shaito A, Motoike T, et al. Effects of the gut microbiota on host adiposity are
modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41. Proc Natl
Acad Sci U S A 2008; 105: 16767–16772.

69. Cani PD, Possemiers S, Van de Wiele T, et al. Changes in gut microbiota control
inflammation in obese mice through a mechanism involving GLP-2-driven improvement of
gut permeability. Gut 2009; 58: 1091–1103.

66. Duca FA, Swartz TD, Sakar Y, Covasa M. Increased Oral Detection, but Decreased Intestinal
Signaling for Fats in Mice Lacking Gut Microbiota. Blachier F, editor. PLoS ONE 2012; 7:
e39748.

70. Cherbut C, Ferrier L, Rozé C, et al. Short-chain fatty acids modify colonic motility through
nerves and polypeptide YY release in the rat. Am J Physiol-Gastrointest Liver Physiol 1998;
275: G1415–G1422.

70. Wichmann A, Allahyar A, Greiner TU, et al. Microbial Modulation of Energy Availability in
the Colon Regulates Intestinal Transit. Cell Host Microbe 2013; 14: 582–590.

This article is protected by copyright. All rights reserved.

 72. Carvalho BM, Guadagnini D, Tsukumo DML, et al. Modulation of gut microbiota by
antibiotics improves insulin signalling in high-fat fed mice. Diabetologia 2012; 55: 2823–
2834.

73. Garner CD, Antonopoulos DA, Wagner B, et al. Perturbation of the Small Intestine Microbial
Ecology by Streptomycin Alters Pathology in a Salmonella enterica Serovar Typhimurium
Accepted Article
Murine Model of Infection. Infect Immun 2009; 77: 2691–2702.

74. Roberfroid MB. Prebiotics and synbiotics: concepts and nutritional properties. Br J Nutr
1998; 80: S197–S202.

75. Nilsson A, Johansson E, Ekström L, Björck I. Effects of a Brown Beans Evening Meal on
Metabolic Risk Markers and Appetite Regulating Hormones at a Subsequent Standardized
Breakfast: A Randomized Cross-Over Study. PLoS ONE 2013; 8: e59985.

76. Parnell JA, Reimer RA. Prebiotic fibres dose-dependently increase satiety hormones and alter
Bacteroidetes and Firmicutes in lean and obese JCR:LA-cp rats. Br J Nutr 2012; 107: 601–
613.

77. Russo F, Linsalata M, Clemente C, et al. Inulin-enriched pasta improves intestinal

permeability and modifies the circulating levels of zonulin and glucagon-like peptide 2 in
healthy young volunteers. Nutr Res 2012; 32: 940–946.

78. Kellow NJ, Coughlan MT, Reid CM. Metabolic benefits of dietary prebiotics in human
subjects: a systematic review of randomised controlled trials. Br J Nutr 2013: 1–15.

79. Høverstad T, Carlstedt-Duke B, Lingaas E, et al. Influence of ampicillin, clindamycin, and

metronidazole on faecal excretion of short-chain fatty acids in healthy subjects. Scand J
Gastroenterol 1986; 21: 621–626.

80. Lewis S, Brazier J, Beard D, Nazem N, Proctor D. Effects of metronidazole and oligofructose
on faecal concentrations of sulphate-reducing bacteria and their activity in human volunteers.
Scand J Gastroenterol 2005; 40: 1296–1303.

81. Rahat-Rozenbloom S, Fernandes J, Gloor GB, Wolever TMS. Evidence for greater
production of colonic short-chain fatty acids in overweight than lean humans. Int J Obes
2014: 38; 1525-31.

82. Aguirre M, Jonkers DMAE, Troost FJ, Roeselers G, Venema K. In Vitro Characterization of
the Impact of Different Substrates on Metabolite Production, Energy Extraction and
Composition of Gut Microbiota from Lean and Obese Subjects. PLoS ONE 2014; 9: 113864.

83. Fava F, Gitau R, Griffin BA, Gibson GR, Tuohy KM, Lovegrove JA. The type and quantity
of dietary fat and carbohydrate alter faecal microbiome and short-chain fatty acid excretion in
a metabolic syndrome “at-risk” population. Int J Obes 2013; 37: 216–223.

84. Mudaliar S, Henry RR, Sanyal AJ, et al. Efficacy and Safety of the Farnesoid X Receptor
Agonist Obeticholic Acid in Patients With Type 2 Diabetes and Nonalcoholic Fatty Liver
Disease. Gastroenterology 2013; 145: 574–582.e1.

This article is protected by copyright. All rights reserved.

 85. Thomas C, Gioiello A, Noriega L, et al. TGR5-mediated bile acid sensing controls glucose
homeostasis. Cell Metab 2009; 10: 167–177.

86. Ridlon JM, Kang DJ, Hylemon PB, Bajaj JS. Bile acids and the gut microbiome: Curr Opin
Gastroenterol 2014; 30: 332–338.
Accepted Article
87. Lefebvre P, Cariou B, Lien F, Kuipers F, Staels B. Role of Bile Acids and Bile Acid
Receptors in Metabolic Regulation. Physiol Rev 2009; 89: 147–191.

88. Out C, Patankar JV, Doktorova M, et al. Gut microbiota inhibit Asbt-dependent intestinal
bile acid reabsorption via Gata4. J Hepatol 2015; DOI 10.1016.

89. Sayin SI, Wahlström A, Felin J, et al. Gut Microbiota Regulates Bile Acid Metabolism by
Reducing the Levels of Tauro-beta-muricholic Acid, a Naturally Occurring FXR Antagonist.
Cell Metab 2013; 17: 225–235.

90. Wostmann BS. Intestinal bile acids and cholesterol absorption in the germfree rat. J Nutr
1973; 103: 982–990.

91. Carulli N, Ponz de Leon M, Loria P, Iori R, Rosi A, Romani M. Effect of the selective
expansion of cholic acid pool on bile lipid composition: possible mechanism of bile acid
induced biliary cholesterol desaturation. Gastroenterology 1981; 81: 539–546.

92. Berr F, Kullak-Ublick GA, Paumgartner G, Munzing W, Hylemon PB. 7 alpha-

dehydroxylating bacteria enhance deoxycholic acid input and cholesterol saturation of bile in
patients with gallstones. Gastroenterology 1996; 111: 1611–1620.

93. Kurdi P, Kawanishi K, Mizutani K, Yokota A. Mechanism of Growth Inhibition by Free Bile
Acids in Lactobacilli and Bifidobacteria. J Bacteriol 2006; 188: 1979–1986.

94. Islam KBMS, Fukiya S, Hagio M, et al. Bile acid is a host factor that regulates the
composition of the cecal microbiota in rats. Gastroenterology 2011; 141: 1773–1781.

95. Devkota S, Wang Y, Musch MW, et al. Dietary-fat-induced taurocholic acid promotes
pathobiont expansion and colitis in Il10-/- mice. Nature 2012; 487: 104–108.

96. Hofmann AF, Eckmann L. How bile acids confer gut mucosal protection against bacteria.
Proc Natl Acad Sci U S A 2006; 103: 4333–4334.

97. Lorenzo-Zúñiga V, Bartolí R, Planas R, et al. Oral bile acids reduce bacterial overgrowth,
bacterial translocation, and endotoxemia in cirrhotic rats. Hepatology 2003; 37: 551–557.

98. Bajaj JS, Heuman DM, Hylemon PB, et al. Altered profile of human gut microbiome is
associated with cirrhosis and its complications. J Hepatol 2014; 60: 940–947.

99. Bauer TM, Schwacha H, Steinbrückner B, et al. Small intestinal bacterial overgrowth in
human cirrhosis is associated with systemic endotoxemia. Am J Gastroenterol 2002; 97:
2364–2370.

This article is protected by copyright. All rights reserved.

 100. Kakiyama G, Pandak WM, Gillevet PM, et al. Modulation of the fecal bile acid profile by gut
microbiota in cirrhosis. J Hepatol 2013; 58: 949–955.

101. Joyce SA, MacSharry J, Casey PG, et al. Regulation of host weight gain and lipid
metabolism by bacterial bile acid modification in the gut. Proc Natl Acad Sci 2014; 111:
7421–7426.
Accepted Article
102. Nieuwdorp M, Vrieze A, Jonker A, et al. Intestinal microbiota is associated with inflamed
visceral adipose tissue. Diabetologia 2012, abstract 55:S1-S538.

103. Amar J, Serino M, Lange C, et al. Involvement of tissue bacteria in the onset of diabetes in
humans: evidence for a concept. Diabetologia 2011; 54: 3055–3061.

104. Moreno-Navarrete JM, Escoté X, Ortega F, et al. A role for adipocyte-derived

lipopolysaccharide-binding protein in inflammation- and obesity-associated adipose tissue
dysfunction. Diabetologia 2013; 56: 2524–2537.

105. Koren O, Spor A, Felin J, et al. Human oral, gut, and plaque microbiota in patients with
atherosclerosis. Proc Natl Acad Sci U S A 2011; 108 Suppl 1: 4592–4598.

106. Van Cromphaut SJ, Vanhorebeek I, d Berghe GV. Glucose metabolism and insulin resistance
in sepsis. Curr Pharm Des 2008; 14: 1887–1899.

107. Amar J, Chabo C, Waget A, et al. Intestinal mucosal adherence and translocation of
commensal bacteria at the early onset of type 2 diabetes: molecular mechanisms and
probiotic treatment. EMBO Mol Med 2011; 3: 559–572.

108. Cani PD, Amar J, Iglesias MA, et al. Metabolic endotoxemia initiates obesity and insulin
resistance. Diabetes 2007; 56: 1761–1772.

109. Mehta NN, McGillicuddy FC, Anderson PD, et al. Experimental Endotoxemia Induces
Adipose Inflammation and Insulin Resistance in Humans. Diabetes 2010; 59: 172–181.

110. Ortiz S, Zapater P, Estrada JL, et al. Bacterial DNA Translocation Holds Increased Insulin
Resistance and Systemic Inflammatory Levels in Morbid Obese Patients. J Clin Endocrinol
Metab 2014; 99: 2573-85.

111. Membrez M, Blancher F, Jaquet M, et al. Gut microbiota modulation with norfloxacin and
ampicillin enhances glucose tolerance in mice. FASEB J Off Publ Fed Am Soc Exp Biol
2008; 22: 2416–2426.

This article is protected by copyright. All rights reserved.

 Figure legend
Accepted Article
Intestinal
epithelium

Dietary Intestinal
fibres lumen
Primary
bile acids
SCFA

Stimulation
of immune cells by Secondary
bacteria or their products bile acids
LPS
Enterocyte
energy substrate

Intestinal
epithelium
Induction of Nuclear FXR Entero endo- systemic energy
inflammation activation crine cells substrate following
upon TLR synthezising absorption
activation gut hormones
GLP-1, PYY,
GLP-2 etc.

Effects on ↑ Insulin ↑ Bile acid Alt. lipid ↑ Intestinal ↑ Insulin ↑ Satiety ↑ Caloric
resistance synthesis metabolism integrity secretion contribution
metabolism

Figure 1. A simplified overview of three pathways through which the gut microbiota can influence
bodyweight regulation and glucose metabolism. Importantly, antibiotics treatment may affect or abrogate all
three pathways.
Short-chain fatty acids (SCFA), lipopolysaccharide (LPS), glucagon-like peptide-1 (GLP-1), glucagon-like
peptide-2 (GLP-2), peptide YY (PYY), farnesoid X receptor (FXR), toll-like receptor (TLR).

This article is protected by copyright. All rights reserved.

 Table 1 Observational studies investigating the association of antibiotic exposure with bodyweight, obesity

or diabetes. Exposure (exp), antibiotics (abx), associate (ass), body mass index (BMI).
Accepted Article
Author, Method, population and country of origin Main association with bodyweight,
reference obesity or diabetes
Mueller Prospective study of child/mother cohort, New York Prenatal exp to abx ass childhood
(13) (n=436) obesity
Mor Retrospective population-based study of school Prenatal exp to abx ass childhood
(14) children, Denmark (n=9,886) overweight
Ajslev Prospective longitudinal study of children from the Exp to abx in infancy not associated
(17) Danish National Birth Cohort (n=28,354) with childhood overweight (main
analysis)
Trasande Prospective longitudinal study of children from the Exp to abx in infancy not ass with
(16) Avon Longitudinal Study of Parents and Children childhood obesity (main analysis)
Cohort (ALSPAC), UK (n=11,532)
Bailey Retrospective cohort study of children, US Exp to abx in infancy ass childhood
(18) (n=65,480) obesity
Azad Nested case-control study of children from a Exp to abx in infancy ass childhood
(20) Canadian birth cohort (n=723) overweight
Murphy Population-based cross sectional study of children Exp to abx in infancy ass with
(19) from 18 diff. countries (n=74,946) childhood BMI
Saari Population-based study of children from a Finnish Exp to abx in infancy ass childhood
(21) birth cohort (n=12,062) BMI and height
Thuny Case-control study of patients admitted due to Abx treatment ass with increases in
(26) suspicion of infective endocarditis, France (n=96) BMI
Angelasakis Case-control study of patients treated with long-term Abx treatment ass with increased
(27) abx for Q-fever endocarditis (cases), France (n=82) weight gain but also weight loss
Francois Prospective cohort study of adults referred for Successful abx eradication of H.
(28) routine upper endoscopy, US (n=69) pylori ass with increases in BMI
Boursi Population-based case-control study, UK Exp to abx ass development of
(34) (n=1,023,578) diabetes (type 1 and 2 combined)
Mikkelsen Nationwide, register-based case-control study, Exp to abx ass development of type 2
(35) Denmark (n=1,534,511) diabetes

This article is protected by copyright. All rights reserved.

 Table 2 Interventional studies investigated the effect of antibiotics on bodyweight or glucose metabolism.
Accepted Article
Antibiotics (abx), placebo (pcb), week (w), day (d)

Study, Design Participants Intervention Effect on bodyweight or glucose

reference metabolism
Lane RCT Unselected H.pylori Abx H.pylori Increases in bodyweight and
(29) positive adults, eradication therapy vs prevalence of obesity in
(n=1558) pcb eradicated group
Vrieze RCT Obese males with 7-d abx treatment with Decreases in peripheral insulin
(37) metabolic syndrome vancomycin vs sensitivity following vancomycin
(n=20) ampicillin but not ampicillin
Reijnder RCT Obese males with 7-d abx treatment with No change in insulin sensitivity
(38) impaired glucose vancomycin, or glucose tolerance following
tolerance (n= 57) ampicillin or pcb vancomycin or amoxicillin vs pcb
Pierce RCT Navy recruits 7-w abx treatment with Increased weight gain in abx
(30) (n=330) aeromycin, penicillin treated groups compared to pcb
or pcb
Mikkelsen Inter- Glucose tolerant 4-d broad-spectrum No changes in postprandial
(31) vention young adults (n=12) abx treatment glucose tolerance, small increase
study in bodyweight following 180 d

This article is protected by copyright. All rights reserved.