Best Practice & Research Clinical Gastroenterology 31 (2017) 579e588

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Best Practice & Research Clinical Gastroenterology

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11

The interaction between smoking, alcohol and the gut microbiome

Gabriele Capurso*, Edith Lahner
Digestive and Liver Disease Unit, Sant’Andrea Hospital, Faculty of Medicine and Psychology, Sapienza University of Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The gastrointestinal microbiome is a complex echosystem that establishes a symbiotic, mutually bene-
Received 20 September 2017 ficial relation with the host, being rather stable in health, but affected by age, drugs, diet, alcohol, and
Received in revised form smoking. Alcohol and smoking contribute to changes in the stomach and affect H pylori-related disorders
17 October 2017
including the risk of gastric cancer. In the small intestine and in the colon alcohol causes depletion of
Accepted 20 October 2017
bacteria with anti-inflammatory activity, eventually resulting in intestinal damage with “leaky gut”.
These changes contribute to hepatic damage in both alcoholic and non-alcoholic liver disease and have
Keywords:
been associated with other disorders. Lactobacillus GG and A. muciniphila exert a protective effect in this
Gastric microbiota
Gastric atrophy
setting. Smoking leads to modifications of the gut microbiome linked with a protective effect toward
Gastric cancer ulcerative colitis and deleterious for Crohn's disease. The exact cause-effect relation between alcohol and
Helicobacter pylori smoking and changes of the gastrointestinal microbiome needs further exploration with high throughput
Alcohol methodologies, and controlled studies are necessary to define the role of microbiome modulation on the
Smoking immune response and systemic activation of pro-inflammatory pathways.
Intestinal microbiota © 2017 Elsevier Ltd. All rights reserved.
Dysbiosis
Liver
Inflammatory bowel disease

1. Introduction diet, alcohol, and smoking [4].

As defined by Quigley EMM, the microbiota refers to the
assemblage of microorganisms (including also others than bacteria)
present in a defined environment, while the microbiome comprises
the full complement of microorganisms, such as bacteria, viruses,
fungi, and protozoa, together with their genes and genomes in a
given locus [1]. The gastrointestinal microbiome is a complex
echosystem of 10e100 trillion microorganisms, of which the ma-
jority are bacteria in the gut, but also including virus and fungal
species, that develops immediately after birth depending on mul-
tiple factors among whom diet and the host genotype seem the
most important ones [2].
The interaction between the microbiome and the host is a
symbiotic, mutually beneficial one.
Data from the Human Microbiome Project Consortium [3] hel-
ped characterizing the microbiome composition and function in
terms of genomics, trascriptomics and metabolomics. In adults the
microbiome tends to become rather stable in health, but is affected
by a number of factors such as age, drugs (especially antibiotics),
* Corresponding author. Digestive and Liver Disease Unit, S. Andrea Hospital,
Sapienza University of Rome, Via di Grottarossa 1035, 00189 Rome, Italy.
E-mail address: gabriele.capurso@gmail.com (G. Capurso).
The most common of the bacterial groups are the gram-negative
Bacteroidetes and the gram-positive Firmicutes, the largest phylum,
that includes several Lactobacillus strains used as probiotics.
In the past decades, it has become clear that the microbiome is
not a passive victim in many pathological processes, but its modi-
fication often play a contributive or causative role in pathophysio-
logical processes [5].
The present review will present the available evidence
regarding the interaction between smoking, alcohol and the gut
microbiome. The effects of alcohol and smoking on the microbiome
of either the upper or the lower gastrointestinal tract will be pre-
sented separately, highlighting key points and area for further
research.
1.1. What do we know about the gastric microbiome?
From a physiological point of view, the stomach may be
considered a defensive barrier against orally ingested microor-
ganisms, as it functions to inactivate ingested microorganisms to
prevent them from reaching the intestine [6]. The observation that
reduced gastric acid secretion increases the susceptibility toward
infection [6,7] supports this concept. The gastric environment is
hostile and difficult to colonize, mainly due to its acidic pH and

https://doi.org/10.1016/j.bpg.2017.10.006
1521-6918/© 2017 Elsevier Ltd. All rights reserved.
580 G. Capurso, E. Lahner / Best Practice & Research Clinical Gastroenterology 31 (2017) 579e588
proteolytic enzymes, and the stomach has therefore been consid-
ered to be a nearly sterile organ. The stomach displays a much
lower microbial load (102-104 colony-forming units [CFU]/ml of
bacteria) than the intestine (104e1012 CFU/ml) [8,9]. Some bacteria
adapt to these unfriendly conditions, i.e. obligate or facultative
acidophiles able to live at pH < 4.0 [10].
In 1983, the discovery of Helicobacter pylori (Hp), a Gram-
negative bacterium responsible of gastric inflammation eventu-
ally leading to peptic ulcer, MALToma or adenocarcinoma, drasti-
cally changed this view. Meanwhile, Hp is certainly the most
thoroughly investigated component of the gastric microbiota [11].
Compared to other gastrointestinal districts, knowledge on
gastric microbiota and its role in human health and disease is
limited. The development of new high-throughput sequencing
methods and technologies as microbiomics and metagenomics
made feasible the investigation of non-cultured microorganisms
[12]. Parallel to this, the wide diffusion of gastrointestinal endos-
copy made easier the accessibility of the stomach to obtain bioptic
mucosal or gastric juice samples for molecular analysis.
How to investigate the gastric microbiota: from culture to
culture-independent molecular methods.
The presence of gastric microorganisms other than Hp was at
first investigated by using conventional methods, such as histology
of gastric mucosal biopsies and the culture of gastric juice or gastric
mucosa [13]. In contrast to animal studies usually using the entire,
aseptically removed stomach, the sample collection in humans is
more complex. Gastric mucosal samples are generally obtained by
biopsies during gastroscopy, but the contamination of the biopsy
channel with oral or throat microorganisms cannot be excluded,
potentially leading to distortion of microbial composition results
given the low gastric bacterial load [11]. Moreover, data are lacking
about microbial differences between different mucosal layers in
humans; microbiota analyses from gastric biopsies represent a
bacteria mix in the luminal fluid, upper mucus layer (non-Hp), and
juxtaepithelial mucus (mainly Hp) [11]. Instead, gastric juice may
be sampled in a sterile manner by using a bull-nosed capsule,
double or triple nastrogastric tube systems, or flexibile silicone
sampling probes [14,15].
By culture, the most represented bacteria in the stomach of
healthy subjects were found to be Clostridium sp, Lactobacillus sp,
and Veillonella sp [14]. The main advantage of the culture-based
approach toward culture-independent methods is the selective
detection of viable bacteria. Unfortunately, the vast majority (~80%)
of microbial species are not cultivable [16].
The more recent culture-independent, molecular approaches
allowed to give new insights in the gastric microbiota composition
and depend on DNA-based approaches, either relying on whole-
genome information or focusing on the 16S rRNA gene as stan-
dard phylogenetic marker [11,17]. First, the bacterial diversity of the
gastric microbial ecosystem was evidenced by more traditional
DNA fingerprinting techniques, such as denaturating gradient gel
electrophoresis (DGGE), temporal gradient gel electrophoresis
(TTGE) or terminal restriction fragment length polymorphism
(TRFLP) of polymerase chain reaction (PCR)-amplified 16S rDNA
fragments [18]; more recently, methods like Sanger-sequencing 16S
rRNA libraries, microarrays targeting the 16S rRNA gene and high-
throughput sequencing methods (454, Illumina, and Ion Torrent)
have been developed [11,12]. These latter methods allow a
considerably higher sequencing coverage than Sanger sequencing
of 16S clone libraries and a much better phylogenetic resolution
than fingerprinting methods, permitting the identification of un-
expected or previously unknown bacteria, not detectable by
microarray approaches [11,19].
A complete view of the overall microbial composition of a def-
inite ecosystem may thus be provided by these innovative
molecular approaches, but with the limit that they are unable to
differentiate living and dead microorganisms [20]. The acquisition
of functional microbiota data requires the sequencing of the com-
plete microbial community DNA by shotgun metagenomics
methods and subsequent matching of the obtained sequences to
the known functional genes available on datasets [9]. Due to the
low bacterial load in the stomach, the application of these methods
may result technically difficult. The techniques used to analyze the
human microbiota are thoroughly reviewed amongst others by
Schulz C et al. [21].
1.2. Who are the bugs in the stomach? what is the composition of
the gastric microbiota?
The major component of the gastric microbiota in more than
half of the world population is Hp, a spiral-shaped member of the
Gram-negative Epsilonproteobacteria [22]. Hp has been classified as
a definitive (class I) carcinogen by the World Health Organization
(WHO). The mucus layer of the stomach is the natural habitat of Hp;
the majority of Hp organisms swim within the mucus layer pro-
pelled by a bundle of rotating flagella [23]. Hp infection profoundly
affects gastric physiologic functions, which in turn may affect the
properties of gastric mucosa to be colonized by other bacteria [24].
Whether Hp infection in humans is able to influence the compo-
sition of the gastric microbiota in not fully understood.
Hp infection leads to changes of the intragastric microenviron-
ment possibly creating novel niches for bacterial species. Rodents'
gastric microbiota may be influenced by Hp infection [25], while
the gastric microbiota in mice seems not to be affected [26]. Simi-
larly, in humans no significant differences between phylotype dis-
tribution of gastric microbiota or gastric pH levels by using 16S
rDNA sequencing analysis were observed between Hp-positive and
enegative subjects [27]. A 2008 European study [28] reported a
higher gastric microbiota diversity in Hpenegative compared to
Hp-positive subjects. Maldonado-Contreras et al. [29] observed a
positive correlation between Hp and Spirochaetae and a negative
correlation between Hp and Bacteroidetes, Chloroflexi, Cianobacteria,
Fusobacteria, Plactomycetes, Beta- and Gamaproteobacteria, and
Verrucomicrobia, further showing that 28% of the microbiota
composition variation was explained by Hp status, but 44% was
explained by patients' origin. Geographical differences may play a
role in the diversity of the human gastric microbiota and in in-
teractions variations between Hp and other gastric microbiota
components [13]. In a very recent study Hp eradication increased
bacterial diversity and restored the relative abundance of other
bacteria to levels similar to Hp-negative subjects suggesting that Hp
colonization results in alterations of gastric microbiota, reversible
by antibiotic treatment [30]. Thus, to date, whether Hp acts as key
player or house keeper for other gastric microbes by modulating
the gastric acidity or by other mechanisms stills remains to be
clarified [21].
In the recent years, culture-independent studies provided evi-
dence on the microbial diversity in the human stomach
[8,27,28,31e33]. Some authors reported the gastric microbiota in
healthy, Hp-free gastric mucosa: Zilberstein et al. [14] isolated 18
different bacterial taxa by culture of gastric mucus aspirate; the
most frequent species were Lactobacillus sp., Veillonella sp., Clos-
tridium sp., and Corynebacterium sp. A study based on 454-
pyrosequencing technology (short rRNA gene sequence reads)
[28] was performed on three Hp-negatives and three Hp-positives):
33 phylotypes were found in all three Hp-negative samples, and
most of the prominent phylotypes (e.g. Streptococcus, Actinomyces,
Prevotella and Gemella), were also abundant in the throat suggest-
ing that they were swallowed microorganisms from upstream
microbiota; the majority of the 177 phylotypes found in the
 G. Capurso, E. Lahner / Best Practice & Research Clinical Gastroenterology 31 (2017) 579e588
stomach but not in throat belonged to Proteobacteria; in contrast,
the three Hp-positive samples were totally dominated by this
bacterium (93e97% of reads), corroborating the finding that the
stomach displays a diverse microbiota when Hp is absent [27].
Li et al. [31] investigated the gastric microbiota composition of
Hp negative NSAIDs-off individuals by 16S rRNA gene-libraries from
antral and corporal biopsies and identified as dominant genera
Streptococcus, Prevotella, Neisseria, Haemophilus, and Porphyr-
omonas, accounting together for 70.5% of all sequences, Strepto-
coccus and Prevotella alone accounted for 41% of all sequences. With
regard to gastric topography, the genus Prevotella was more
abundant in the antrum compared to the corpus.
Similarly to previous studies with different methodological ap-
proaches [27,28] Stearns and colleagues [32] by using Illumina
sequencing reported Firmicutes, Actinobacteria, Bacteroidetes, Pro-
teobacteria, and Fusobacteria as the most abundant phyla of the
human stomach. Albeit the microbial research in the human
stomach has been defined to be still in the early stages, current data
show that the stomach microbiota displays a microbial diversity
beyond Hp and seems to be distinguishable from oral, throat, nose,
and distal gut microbiota [8,13], supporting the idea that in healthy
subjects harbor a “core gastric microbiota”. Table 1 gives an over-
view on the reported composition of the gastric microbiota other
than Hp.
1.3. Potential modulators of the gastric microbiota: alcohol and
smoking
Microbiota diversity or composition may be shaped by the host
physiology as well as by environmental factors. Many factors have
been postulated to potentially exert an influence on the gut
microbiota by causing significant shifts of its composition, such as
diet, antibiotics, other drugs, Hp-related inflammation, intra-gastric
pH, as well as smoking and alcohol [33]. For example, the mucus
layer in the stomach is a protective barrier preventing microbial
epithelial colonization and diffusion of gastric acid to the epithelial
surface. The capability to get across the mucus layer highly depends
on bacterial factors (motility, shape and mucus-degrading-ability)
[34], but also on host factors: an increased pH results in lower
viscosity making the mucus layer easier to penetrate for microor-
ganisms [35], thus potentially modulating gastric microbiota. Hp
infection is linked to chronic inflammation, oxidative stress, and
DNA damage [36], potentially influencing the gastric microbiota
composition. An elegant study in wild-type C57BL/6 mice reported
that the entity of Hp-associated inflammation was directly related
Table 1
Composition of the gastric microbiota beyond Helicobacter pylori.
Most frequent phyla/genera Detection technique
Corynebacterium Culture of gastric mucus aspirate
Clostridia
Lactobacillus
Veillonella
Actinomyces 454-pyrosequencing technology (short rRNA gene sequence reads) from gastric biopsies
Gemella
Prevotella
Proteobacteriaa
Streptococcus
Haemophilus 16S rRNA gene-libraries from antral and corporal biopsies
Neisseria
Porphyromonas Prevotella
Streptococcus
Actinobacteria Illumina 16S rRNA sequencing of gastric biopsies
Bacteroidetes, Firmicutes
Fusobacteria Proteobacteria
a
Proteobacteria were detected the stomach only, the other also in the throat; Hp ¼ Helicobacter pylori.
581
to the pre-infection microbiota [37], suggesting that gastric
microbiota variations may have an effect of Hp-associated disease.
An impaired gastric microbiota (dysbiosis) has been identified as a
possible trigger of different gastric conditions as atrophic gastritis
(AG), intestinal metaplasia, and even gastric cancer (GC)
[33,38e40].
The gastric microbiota has long been considered an essential
factor contributing to GC development [41]: The impaired gastric
acid secretion in gastric atrophy results in gastric bacterial over-
growth, increased production of N-nitroso-compounds leading to
DNA damage and epithelial cells methylation, potentially promot-
ing gastric carcinogenesis [42]. This is supported by data showing
increased gastric bacterial counts and enhanced nitrites production
in the stomach during antisecretory treatment [43].
1.3.1. Gastric microbiota modulation and gastric cancer
Yu G et al. [38] showed that lower microbial richness was
significantly associated with lower pepsinogen I/II ratio, a sero-
logical marker of gastric atrophy predisposing to GC, supporting the
potential role of the gastric microbiota in GC risk. In GC patients, the
gastric microbiota was dominated by different species of the genera
Streptococcus, Lactobacillus, Veillonella, Prevotella, and Haemophilus
[44]. Veillonella and Haemophilus parainfluenzae had a higher ca-
pacity in nitrate than nitrite reduction, thus increasing nitrite
accumulation in the gastric juice [45].
A recent Korean study analyzed the gastric microbiota of Hp-
positive and -negative GC patients compared to controls by bar-
coded 454 pyrosequencing of the 16S rRNA gene and showed a
higher composition of Streptococcus pseudopneumoniae,
S. parasanguinis, and S. oralis in the Hp-negative cancer group [46],
suggesting a possible role of non-Hp bacteria in the gastric carci-
nogenesis. However, a very recent Swedish study investigated the
composition of the transcriptionally active microbial community
and the Hp gene expression by using metatranscriptomic RNA
sequencing of stomach biopsies from individuals with different Hp
infection status and pre-malignant tissue changes observing that
Hp completely dominates the microbiota not only in infected in-
dividuals, but also in most individuals classified as Hp-uninfected
using conventional methods, and that Hp abundance is positively
correlated with presence of Campylobacter, Deinococcus, and Sul-
furospirillum [47]. This study clearly strengthens how functional
analysis of the gastric microbiota may provide new insights into
how bacteria respond in vivo to variations in the stomach micro-
environment and how much we have still to learn about its in-
teractions and modulators.
Subjects Reference
Healty volunteers [14]
Hp-negative subjects [27]
Hp-negative NSAIDs-off subjects [31]
Healthy volunteers [32]
582 G. Capurso, E. Lahner / Best Practice & Research Clinical Gastroenterology 31 (2017) 579e588
1.3.2. Gastric microbiota modulation and alcohol
Alcohol may be considered a further modulator of gastric
microenvironment. Attention has been paid to the harmful conse-
quences of alcohol in the stomach. Both acute and chronic alcohol
consumption produce harmful effects in the stomach by multiple,
complex mechanisms related to direct contact of ethanol or its
metabolite acetaldehyde as well as by non-alcoholic components of
alcoholic beverages: (i) gastric motility impairment; (ii) alteration
of gastric acid output, and (iii) direct mucosal damaging [48,49].
Ethanol solutions exert an inhibitory, not dose-dependent effect on
gastric emptying, and alcoholic beverages produced by fermenta-
tion (beer and wine) are emptied even more slowly than their
corresponding ethanol concentrations, presumably due to addi-
tional inhibitory factors in the non-alcoholic ingredients [50]. Fer-
mented alcoholic drinks are potent stimulators of gastric acid
output, and succinic acid and maleic acid have been reported as the
responsible substances [51]. In contrast, highly concentrated alco-
holic beverages produced by distillation (whiskey and cognac) do
not stimulate gastric acid secretion; interestingly, alcoholic bever-
ages produced by fermentation followed by distillation (i.e. aperi-
tifs and hard liquors) do not stimulate acid secretion as well [52].
Alcohol has been associated with a breakdown of gastric barrier
thus contributing to the toxic damage of alcohol to gastric mucosa
and alterations in mucosal defense [53]. Ethanol may directly and
dose-dependently impair the gastric mucosal barrier; the acidifi-
cation of the mucosal cells and ethanol itself induce release of in-
flammatory and vasoactive substances, leading to ischemia and
mucosal damage [52]. Therefore, analogously to tobacco smoking,
acute or chronic use/abuse of alcohol may be postulated to signif-
icantly alter the gastric microenvironment in turn affecting the
gastric microbiota composition.
With regard to Hp, an alcohol intake lower than 75 g weekly has
been reported to protect against active infection [54]. Another
study reported a lower risk of Hp infection in subjects consuming
between 3 and 6 units of wine or beer weekly compared to those
not drinking (OR 0.89, 95% 0.90e0.99; OR 0.83, 95%CI 0.75 to 0.91)
[55]. A recent dose-response meta-analysis showed an inverse
relationship between alcohol intake and Hp infection (OR 0.78,95%
CI 0.69 to 0.89), regardless of gender, age, geographic areas,
detection methods or beverage [56], thus confirming the results
from former studies.
Alcohol consumption has been reported to be positively asso-
ciated with successful eradication treatment: treatment failure was
significantly more frequent in non-consumers (29.9%) than in
consumers (12.2%) (OR 3.24) [57]. To date it is not understood
whether alcohol-related alterations of the gastric microenviron-
ment and possible subsequent changes of the gastric microbiota
may be linked to this observation.
In contrast to the observed protective effects of alcohol on Hp
infection, alcohol intake was reported being a risk factor for GC
[58e60]: Low alcohol intake was negatively associated with GC risk
(HR 0.74) [61]. This association was stronger only in heavy alcohol
consumers (4 drinks daily) (RR 1.20) [62]. A more recent huge
Japanese study reported a significant association with GC risk and
different levels of alcohol intake (HR 1.39) [63]. Similarly to
smoking, alcohol intake has been reported as being associated with
Hp eradication failure [64].
Ethanol is metabolized by alcohol dehydrogenases (ADH),
catalase or cytochrome P450 2E1 to acetaldehyde which is then
further oxidized to acetate by aldehyde dehydrogenase (ALDH)
[65]. Acetaldehyde induces DNA damage in the gastrointestinal
mucosa [65], classified as class 1 carcinogen to humans by the WHO
[66]. Several mechanisms have been suggested to delineate alcohol
consumption and cancer development. Ethanol itself is not carci-
nogenic, but data suggest that acetaldehyde and ROS have
genotoxic or tumor promoting effects. In AG, a predisposing con-
dition for GC characterized by impaired gastric acid secretion and
alterations of the gastric microbiota [40,67], ethanol metabolism by
the gastric microbiota may lead to high intragastric acetaldehyde
production [68]: the acetaldehyde production from gastric juice
samples from AG patients incubated with 1% ethanol has been re-
ported to be 7.6-times higher compared to control samples [69].
Thus, alcohol consumption may exert effects on the gastric
microenvironment and this may, in turn, lead to potential alter-
ations of the gastric microbiota composition, including Hp. The
magnitude and the modalities with which alcohol intake may
modulate Hp infection and the gastric microbiota remain a subject
open for investigation, as specific studies on the gastric microbiota
beyond Hp are lacking.
1.4. Gastric microbiota modulation and smoking
N-nitroso compounds, consisting of N-nitroamines and N-
nitroamides, are potent carcinogens to which humans are exposed
from diet and smoking. Increased exposure to these exogenous N-
nitroso compounds has been linked to an increased incidence of GC,
albeit an important role has been ascribed to the endogenous N-
nitroso compounds [70]. A 2008 systematic review provided
summary RR estimates of 1.62 in males and 1.20 in females for GC in
current smokers compared to never smokers, classifying smoking
as one the most important behavioral risk factor for GC [71]. More
recently, data from the Stomach Cancer Pooling (StoP) Project
confirmed the detrimental effect of cigarette smoking on the risk of
GC reporting a RR 1.25 for current cigarette smokers compared to
never smokers [72].
Another proposed mechanism by which smoking may lead to
negative effects on health, as cancer and periodontitis, may be
through microbiome alterations [73]. Vogtman E et al. showed by a
16S rRNA microarray that current smokers had an increased
within-participant-diversity for a 10 species increase, the OR for
current smoking was 1.29; Dialister invisus and Megasphaera
micronuciformis were more commonly detected in current smokers
compared to never smokers [74].
Among host-related factors, smoking has been studied as a
possible risk factor for Hp eradication failure: a meta-analysis re-
ported an OR of 1.95 for eradication failure in smokers compared to
non-smokers [75]. A very recent Israeli study on >48,000 subjects
confirmed that eradication failure (first-line treatment for Hp
infection) was positively associated with current smoking (OR 1.15),
together with female gender (OR 1.20) and low socioeconomic
status (OR 1.24) [76]. Whether the significant association between
the likelihood of unsuccessful first-line treatment for Hp infection
and smoking status may be linked to an altered gastric microbiome
remains to be investigated.
Table 2summarizes the supposed mechanisms and the
described modulating effects of alcohol and smoking on the gastric
microbiota.
1.5. Effects of alcohol on the microbiome of the lower
gastrointestinal tract
The association between alcohol consumption and changes in
intestinal microbiome has been investigated extensively in the past
decade and there is sufficient evidence suggesting that both acute
and chronic exposure to alcohol determine specific modifications of
the microbiome composition, bacterial overgrowth, and disruption
of the mucosal barrier function [77] (see Table 3). Many of these
studies were conducted in animal models with liver disease, as it
has become clear that alcohol-induced intestinal dysbiosis alters
the homeostasis between the gut and the liver and plays a central
 G. Capurso, E. Lahner / Best Practice & Research Clinical Gastroenterology 31 (2017) 579e588 583

Table 2
Alcohol and smoking as potential modulators of the gastric microbiota.

Gastric microbiota Proposed mechanisms Alteration of the gastric microbiota

modulators

Alcohol Impairment of gastric motility Inverse relationship between alcohol intake and Hp infection
Alteration of gastric acid output Alcohol consumption is positively associated with successful Hp
Breakdown of gastric mucosal barrier eradication treatment
Production of acetaldehyde Alcohol intake is associated with higher gastric cancer risk; in atrophic
gastritis, higher acetaldehyde levels and gastric microbiota alterations
Other gastric microbes: data are lacking
Smoking Alteration of gastric microenvironment: increased exposure to N- Smoking is associated with a higher risk of Hp eradication failure
nitroso compounds which are potent carcinogens inducing DNA Other gastric microbes: data are scanty
damage and epithelial cells methylation, possibly enhanced by impaired Smoking is associated with higher gastric microbial diversity Smoking is
gastric acid secretion leading in turn to increased nitroso-compounds associated with a higher risk of gastric cancer
production due to gastric bacterial overgrowth In GC patients, gastric microbiota dominated by Streptococcus,
Lactobacillus, Veillonella, Prevotella

Hp ¼ Helicobacter.

Table 3
Effects of alcohol on intestinal microbiome composition and associated disorders.

Animal Models Human Studies Intestinal damage Associated Disorder(s)

Phyla (Genera) Firmicutes (Lactococci, Bacteroidetes [88,91,92], Bacterial overgrowth, leaky gut, Intestinal inflammation, ASH,
Depleted by Alcohol Pediococci, Lactobacilli) [78,80], Proteobacteria changes of mucus composition, Klebsiella pneumoniae
A muciniphila [84] (Enterobacteriaceae) [91,92] bacterial translocation. pulmonary infection.
Firmicutes (Lachnospiraceae,
Ruminococcaceae) [89,91],
Verrucomicrobia (A muciniphila)
Phyla/genera increased Verrucomicrobia, Bacteroidetes Proteobacteria
by alcohol Actinobaceria [78,80,89,91] (Enterobacteriaceae)
Bacteroides, Clostridium
[88,89,91]
Genera associated with LGG [79,80], A muciniphila [85] LGG [92] e
recovery of damage

LGG ¼ Lactobacillus rhamnosus GG, ASH ¼ alcoholic steatohepatitis.

role in the development of alcoholic liver disease. In this view, the with faeces of control animals fed with alcohol, intestinal and he-
microbiome plays the role of an important mediator of a number of patic inflammation increases, suggesting that the pro-
negative effects of alcohol. However, it has also been demonstrated inflammatory effect of alcohol is mediated by the intestinal
that bacterial phyla are able to produce alcohol themselves, such as microbiome [81].
Proteobacteria, are increased in subjects with metabolic alterations Chronic exposure to alcohol also modifies the composition of
and non-alcoholic steatohepatitis (NASH), possibly supporting the intestinal mucus. Interestingly, MUC2 knock out animals are less
hypothesis of a direct role of the microbiome in determining NASH. prone to develop bacterial overgrowth, bacterial translocation and
At any rate, manipulation of the microbiome is an intriguing ther- intestinal inflammation in response to exposure to alcohol, sug-
apeutic possibility for alcohol-related disorders. gesting that the interaction between alcohol, the mucosal layer and
microbiome are complex [82]. An interesting bacterial species in
this setting is Akkermansia muciniphila, a member of the phyla
1.5.1. Studies in animal models
Verrucomicrobia, with growing data supporting potential anti-
There are a number of studies in animal models in which
inflammatory properties [83]. A muciniphila exerts an anti-
exposition to alcohol causes ‘leaky gut’ with decreased levels of
inflammatory action, and its depletion is an early event in
bacteria with “anti-inflammatory activity” such as Firmicutes (Lac-
alcohol-fed animals [84]. In mice treated with alcohol, when A
tococcus, Pediococcus, Lactobacillus and the Leuconostoc genera) and
muciniphila is administered in a preventive setting, all markers of
increased levels of Verrucomicrobia and Bacteroidetes [78] in the
intestinal (gut barrier function and mucus thickness) and liver
cecum. These changes seem to occur after 3 weeks of exposure to
damage are reduced [85].
alcohol. In another study, rats fed with alcohol for 10 weeks showed
Taken together, these studies strongly suggest that alcohol
modifications of the microbiome in the colonic but not in the ileal
causes significant changes in the composition of the colonic
mucosa, and pre-treatment with either a probiotic (Lactobacillus
microbiome in animal models, which in turn causes pro-
GG, LGG) or a prebiotic (oats) prevented these changes [79].
inflammatory changes, leaky gut and bacterial translocation asso-
Similar results have been confirmed by others, with data sug-
ciated with liver disease [86].
gesting that alcohol causes an increase of Proteobacteria and Acti-
The liver is not the only organ distant from the gut that has been
nobaceria phyla, and a decrease of Firmicutes with these changes
associated with deleterious effects of intestinal dysbiosis due to
being restored by treatment with LGG [80].
alcohol. As alcohol is a risk factor for pulmonary infections,
Germ-free animals are an interesting model to investigate the
Samuelson and colleagues investigated the effect of alcohol-
role of alcohol on intestinal microbiota. When germ-free rats are
induced dysbiosis on the susceptibility to Klebsiella pneumoniae
fed with alcohol for seven days the induction of pro-inflammatory
infection in an animal model [87]. When alcohol-naïve rats were
cytokines and the liver inflammation are less pronounced
recolonized with microbiota from alcohol-fed animals, there was
compared to those seen in control animals with normal micro-
an increased risk of K. pneumoniae infection and related damage,
biome. Furthermore, when germ-free animals are transplanted
584 G. Capurso, E. Lahner / Best Practice & Research Clinical Gastroenterology 31 (2017) 579e588
associated with impaired immunological defense. These findings
point out toward a wider role of alcohol-related intestinal dysbiosis
with potential effects on the immunological response and conse-
quences that are not limited to the digestive system.
1.5.2. Studies in human
There is also a growing body of evidence form studies in humans
supporting the association between alcohol consumption, dysbio-
sis and eventually intestinal and hepatic inflammation. Chronic
alcohol consumption leads to an increase in Proteobacteria and a
decrease in Bacteroidetes and increased intestinal permeability [88]
resulting in traslocation of LPS and endotoxins in the bloodstream
which eventually contributes to hepatic damage [89].
Indeed, the microbiome of cirrhotic patients differs from that of
controls, and a “cirrhosis dysbiosis ratio” (CDR) has been proposed
[90].
This index, defined by the ratio between Lachnospiraceae and
Ruminococcaceae which can exert a protective effect and Entero-
bacteriaceae and Bacteroidaceae that are potentially pathogens is
typically lower in cirrhotics, especially in those with an alcoholic
etiology [89,91].
The causal effect of these alteration is unclear. However,
intriguingly, administration of Lactobacillus rhamnosus GG to
cirrhotic patients in a randomized-controlled trial, determines an
increase of Lachnospiraceae and a decrease of Enterobacteriaceae
with consequent reduction of pro-inflammatory cytokines [92].
Although all these data clearly show that alcohol favours dys-
biosis, some alcoholic beverages might exert a positive effect on the
microbiome. This is the case of red wine. In a cross-over study
performed in healthy subjects, gin caused an increase in the Bac-
teroides and Clostridium frequencies while both red wine and de-
alcoholized red wine consumption led to an increase of the
amount of a number of bacterial group including Enterococcus,
Bacteroides and Bifidobacterium [93]. Red wine induced changes
were associated with a parallel reduction of systolic and diastolic
blood pressures, triglyceride, cholesterol, and C-reactive protein.
The results suggest that the effect is mediated by polyphenols
contained in red wine.
A decrease of faecal A. muciniphila when compared with healthy
controls has also been reported in patients with alcoholic steato-
hepatitis (ASH) which seems associated the severity of the liver
damage [85]. A. muciniphila is a key health-promoting bacteria
related with active and healthy lifestyle, as regular physical activity
increases its abundance together with that of Bifidobacterium spp,
R. hominis, and F. prausnitzii [94].
Taken collectively these data demonstrate that specific modifi-
cations of the microbiome occur in association with alcohol intake
in humans, causing intestinal damage and increase in inflammatory
changes, eventually linked with increased hepatic damage. With
this in mind, Llopis et al. designed a study in which germ-free and
conventional mice received microbiome transplants from alcoholic
patients with or without alcoholic hepatitis and investigated
possible different effects of feeding the animals with alcohol related
with this “humanized” microbiome [95]. They found that mice who
Table 4
Effects of smoking on intestinal microbiome composition and associated disorder(s).
Animal Models
Phyla (Genera) Depleted by Bifidobacteria [111],
Smoking Firmicutes
(Lactococcus,Ruminococcus),
Enterobacteriaceae [112]
Phyla/genera increased Clostridium [112],
smoking Lachnospiraceae [113]
received the microbiome of patients with severe alcoholic hepatitis
developed a worse intestinal, damage with increased intestinal
permeability and bacterial translocation, and more prominent in-
flammatory changes in the liver. This result strongly supports the
case of an active role of the microbiome, independently from
alcohol and the host.
Another intriguing aspect regards the existence of a gut-brain
axis that could be modulated by microbiome imbalance due to
alcohol. In an interesting study, Leclercq et al. [96] investigated the
composition of gut microbiome and intestinal permeability and
their association with psychological symptoms of alcohol depen-
dence in 75 subjects with alcohol dependence. They found that
subjects with increased intestinal permeability had higher scores of
depression and anxiety and alcohol craving after 3 weeks of
abstinence, possibly suggesting that the microbiome might be a
potential target to reduce the risk of alcohol use relapse in
alcoholics.
An interesting issue regards the cause-effect relation between
alcohol and microbiome. Indeed, while the above mentioned
studies suggest that alcohol assumption modifies the microbiome
and that their interaction determines a number of pathophysio-
logical changes related with intestinal and liver damage, dysbiosis
itself can lead to ethanol production even in the absence of alcohol
assumption, thus possibly resulting in secondary liver damage.
It has been demonstrated that some bacteria, such as Escherichia
coli can produce up to 0.8 g of ethanol per hour in anaerobic con-
ditions [97]. Interestingly, the presence of Enterobacteriaceae which
under certain circumstances can produce alcohol, is increased in
patients with NASH [98]. In another study ethanol was measured by
gas chromatography in 21 with NASH and in 10 controls [99]. While
NASH patients did not produce more ethanol compared to controls,
its levels were higher in obese women. One might therefore spec-
ulate that a certain type of dysbiosis, typical of obesity and meta-
bolic syndrome, favours increased ethanol production in the gut,
contributing to liver injury in a vicious circle in which increased
ethanol production would in turn cause “leaky gut” and bacterial
translocation eventually causing increased liver damage.
1.6. Smoking and the microbiome in the lower GI tract
Cigarettes produce as much as 4000 individual chemical com-
pounds such as hydrogen, hydrogen cyanide, methane, aniline,
aldehyde, phenols, nicotine and nar containing in turn simple
phenols, cresols, naphthols, pyrene, benzopyrene and hydrocar-
bons and heavy metals such as lead, cadmium, nickel, polonius,
strontium [100]. All these compounds have some kind of delete-
rious effects on different human organs and some of them exert an
effect on the intestinal health and on the microbiome (see Table 4).
1.6.1. Studies in animal models
Animal models offer a good opportunity to analyze the affects of
different compounds derived from cigarette smoking on the in-
testine [101]. However, there is a relative paucity of these kind of
data in the literature. Thomas et al. investigated the effect on
Human Studies Intestinal damage Associated Disorder(s)
Firmicutes, Actinobacteria [115] Anti-inflammatory effect in the Crohn's disease and Ulcerative
colon mucosa and Colitis.
proinflammatory in the ileum;
changes in mucins expression.
Clostridium [114] Bacteroidetes,
Proteobacteria [115]
 G. Capurso, E. Lahner / Best Practice & Research Clinical Gastroenterology 31 (2017) 579e588
colorectal mucosa of subcutaneous nicotine at different doses as
compared to saline, in ferrets. They did not report significant
changes but for the nicotine and cotinine serum levels [102]. Van
Dijk et al. reported the potential anti-inflammatory effect of nico-
tine, as upon its administration the synthesis of pro-inflammatory
cytokines such as interleukin-1 beta and tumor necrosis factor
alpha was completely abolished [103].
The effects of smoking on the large and small bowel mucosa
seem to diverge. It has been demonstrated that both nicotine and
carbone-monoxide do not cause macroscopic or microscopic
damage and decrease inflammation in the colon, while nicotine
worsens jejunitis in experimental models, decreasing PGE2 gen-
eration and increasing NOS activity [104e106]. These data support
the clinical observation of opposite effects of smoking on ulcerative
colitis and Crohn's disease. However, there are data in opposition
with this view, as others found that cigarette smoke (CS) exposure
in a rat model of trinitrobenzene sulfonic acid (TNBS)-induced
colitis worsened the inflammatory damage [107].
Zijlstra and colleagues investigated the effect of different con-
centration of subcutaneous nicotine on the colonic mucosa of
rabbits [108]. The thickness of adherent mucus on rectal mucosa
was significantly reduced by low dose nicotine, but increased by
high doses. Prostaglandins (PG) were reduced with an inverse dose
dependence, as lower doses caused a more significant reduction.
This heterogeneity might be due to the different models, the
different substances (either nicotine or cigarette smoking) and
their dosage and way of administration employed in the above
mentioned studies.
In this context, it is worth reminding that the intestinal micro-
biota is an important scavenger of potentially toxic compounds,
with a number of enzymatic families (azoreductases, nitro-
reductases, b-glucuronidases, sulfatases and b-lyases) affecting the
metabolism of over 30 environmental pollutants. On the other
hand, environmental contaminants alter the composition of the
microbiome [109,110].
It is therefore not surprising that exposure to smoking causes
modifications of the gut microbiome. A decrease of Bifidobacteria
and of proprionic and butirric acid in the cecum has been reported
by Tomoda et al. in rats exposed to smoking [111].
In a similar animal model, even passive smoking increases
Clostridium sp and reduces Firmicutes phylum (Lactococcus and
Ruminococcus sp.) and Enterobacteriaceae [112]
In a more recent study, the effect of chronic exposure to smoking
compared to normal air was evaluated on the mucosa and on the
microbiome composition of rats at different levels of the GI tract
(ileum, cecum, colon) employing the Illumina high throughput
sequencing [113].
Microbial clusters in the cecum and colon differed significantly
in animals exposed to smoking with an important increase of
Lachnospiraceae sp. The mRNA expression of mucins was also
modified by smoking, as Muc2 and Muc3 increased in the ileum,
whereas Muc4 increased in the distal colon of smoke-exposed
mice, while the expression of tight junction genes remained
unchanged.
1.6.2. Studies in humans
The reported increase of Clostridia induced by smoking in rats
seems also indirectly confirmed in humans by an epidemiological
study on 16781 elderly subjects. The authors reported an increased
rate of C. difficile infection in smokers, with odds 33% greater in
former smokers and 80% greater in current smokers when
compared to never smokers [114].
Smoking withdrawal in humans determines profound changes
in the microbiome with increased microbial diversity [115], an in-
crease of Firmicutes and Actinobacteria and a decrease of
585
Bacteroidetes and Proteobacteria. Of course, the observed results
might be due to other uninvestigated factors related with smoking
habit or with its cessation, such as modification of the diet and
increase in body weight. Interestingly, similar changes have been
reported in obese men with increased levels of Firmicutes and
reduced levels of Bacteroidetes [116].
As stated above, the heterogeneous effects of smoking on the
human microbiome at different sites might partially explain the
reduced risk of ulcerative colitis [117] and the increased risk of
Crohn's disease (CD) [118] associated with this habit.
Indeed, in Crohn's disease patients who smoke, the microbial
population and diversity is decreased with reduction in particular
of Collinsella, Enterorhabdus and Gordonibacter [119]. In another
study the decreased levels of Faecalibacterium and smoking are
associated independently with CD recurrence [120].
2. Conclusion remarks
In conclusion, microbiome research has enormously developed
in the last years tempting to move its first steps from the laboratory
into clinical practice. New nucleotide sequencing techniques and
new biocomputational tools techniques have allowed to better
characterize the microbiome of the gastrointestinal tract. Among
many factors that contribute to the modulation of the gastroin-
testinal microbiome, the intake of alcohol and smoking seem
relevant. The interactions between alcohol and smoking, the gastric
microbiome and the host remain enigmatic. This holds true also for
the role of H. pylori which might act as a key player or house keeper
for other GI microbes by modulating gastric acidity. As stated by
Quigley EMM [1], for the real clinical applicability of gut micro-
biome research at least three essential conditions need to be ful-
filled: (i) we need to know what the normal microbiota is like, (ii)
we need to accurately and reliably define an altered/abnormal
microbiota, and (iii) we need to establish a biologically plausible
and clinically meaningful relationship between a certain micro-
biome profile and a certain disease [1]. This holds true especially for
the gastric microbiota, as to date the composition of the normal
“core” gastric microbiota is still not completely defined. The precise
role of potential modulators of gastric microbiota, such as smoking
and alcohol, still needs to be clarified, but current available data
suggest a key role in gastric diseases, in particular as risk modulator
for gastric cancer. As far as regards the interaction between alcohol
and smoking and the intestinal microbiome, there is sufficient ev-
idence suggesting that alcohol causes depletion of certain microbes
and increase of others, and that the resulting dysbiosis, coupled
with leaky gut causes increased inflammatory changes in the in-
testine and liver. These alterations seem particularly relevant for
the pathogenesis of alcoholic liver disease. Specific bacteria such as
Lactobacilli and A. muciniphila that are depleted by alcohol seem
able to exert a significant anti-inflammatory effect in this setting.
Interestingly, dysbiosis itself in non-alcoholic steatohepatitis in-
crease alcohol production in a potential vicious circle. Alcohol-
induced modifications of the gut microbiome also seem associ-
ated with other non-gastrointestinal disorders, including pneu-
monia and behavioral patterns. The role of smoking in determining
modifications of the gut microbiome has been investigated spe-
cifically in the context of inflammatory bowel disease, and links
with the long-standing clinical observation of smoking being pro-
tective toward ulcerative colitis and deleterious for Crohn's disease.
In the next few years the puzzle of knowledge on this topic will
certainly become more complete.
However, in this complex research field, many confounders
come into play. Not least because the microbiota-gut-brain axis is
bidirectional, as shown by the effects of stress on gut physiology,
immune function and gut microbiota composition [121]. This may
586 G. Capurso, E. Lahner / Best Practice & Research Clinical Gastroenterology 31 (2017) 579e588
be particular true for life style habits as smoking and alcohol con-
sumption related with psychological and social factors, which
might in turn influence the gastric microbiota.
Practice Points:
 Alcohol and smoking deeply modify the microbiome of
both the upper and the lower gastrointestinal tract
 The resulting dysbiosis is related with immune response,
“leaky gut” and systemic consequences contributing to
gastric, intestinal, liver and distant organs damage
 Modulation of the gastrointestinal microbiome with spe-
cific bacteria, such as LGG or A muciniphila has the po-
tential to modify disease course of alcoholic and non-
alcoholic liver disease, inflammatory bowel disease and
other disorders
Research agenda:
 The exact cause-effect relation between alcohol and
smoking and changes of the gastrointestinal microbiome
needs further exploration with high throughput method-
ologies, especially in the upper GI tract.
 Detailed controlled studies are necessary to define the
role of microbiome modulation on the immune response
and systemic activation of pro-inflammatory pathways
 The role of the gut-brain axis is also a potential interesting
area for research as dysbiosis can influence the central
nervous system response
Conflict of interest
None.
Sources of funding and conflicts
The authors declare no supporting funds and no conflicts of
interest.
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