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RESEARCH ARTICLE
Effect of the proton pump inhibitor omeprazole on the
gastrointestinal bacterial microbiota of healthy dogs
Jose F. Garcia-Mazcorro
1
, Jan S. Suchodolski
1
, Katherine R. Jones
2
, Stuart C. Clark-Price
2
,
Scot E. Dowd
3
, Yasushi Minamoto
1
, Melissa Markel
1
,Jo
¨rg M. Steiner
1
& Olivier Dossin
2
1
Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, Texas A&M University, College Station, TX, USA;
2
Department of
Veterinary Medicine, University of Illinois, Urbana-Champaign, IL, USA; and
3
Molecular Research LP (MR DNA), Shallowater, TX, USA
Correspondence: Olivier Dossin, National
Veterinary School of Toulouse, Department
of Clinical Sciences and Clinical Research
Unit, 23 Chemin des Capelles BP 87614,
31076 Toulouse Cedex 3, France. Tel.:
+33 561 193830; fax: +33 561 193203;
e-mail: o.dossin@envt.fr
Received 31 August 2011; revised 2 February
2012; accepted 6 February 2012.
Final version published online 12 March
2012.
DOI: 10.1111/j.1574-6941.2012.01331.x
Editor: Julian Marchesi
Keywords
Omeprazole; 454-pyrosequencing;
microbiota; stomach; duodenum; canine.
Abstract
The effect of a proton pump inhibitor on gastrointestinal (GI) microbiota was
evaluated. Eight healthy 9-month-old dogs (four males and four females)
received omeprazole (1.1 mg kg
1
) orally twice a day for 15 days. Fecal sam-
ples and endoscopic biopsies from the stomach and duodenum were obtained
on days 30 and 15 before omeprazole administration, on day 15 (last day of
administration), and 15 days after administration. The microbiota was evalu-
ated using 16S rRNA gene 454-pyrosequencing, fluorescence in situ hybridiza-
tion, and qPCR. In the stomach, pyrosequencing revealed a decrease in
Helicobacter spp. during omeprazole (median 92% of sequences during admin-
istration compared to >98% before and after administration; P=0.0336),
which was accompanied by higher proportions of Firmicutes and Fusobacteria.
FISH confirmed this decrease in gastric Helicobacter (P<0.0001) and showed
an increase in total bacteria in the duodenum (P=0.0033) during omeprazole.
However, Unifrac analysis showed that omeprazole administration did not sig-
nificantly alter the overall phylogenetic composition of the gastric and duode-
nal microbiota. In feces, qPCR showed an increase in Lactobacillus spp. during
omeprazole (P<0.0001), which was accompanied by a lower abundance of
Faecalibacterium spp. and Bacteroides-Prevotella-Porphyromonas in the male
dogs. This study suggests that omeprazole administration leads to quantitative
changes in GI microbiota of healthy dogs.
Introduction
The secretion of gastric acid is one of the first defense
mechanism in the body to avoid the introduction of
potentially harmful infectious agents into the intestinal
tract. Gastric acid is secreted by the parietal cells and is
regulated by complex paracrine, endocrine, and neural
pathways (Yao & Forte, 2003).
Proton pump inhibitors (PPIs) are drugs of widespread
therapeutic use in human and veterinary medicine. PPIs
inhibit the secretion of gastric acid by blocking the H
+
/
K
+
-ATPase in gastric parietal cells (Sachs et al., 1995). In
humans, a recent retrospective study of 125 patients
showed that advanced age, low serum albumin concentra-
tions, and concomitant use of PPIs were significant risk
factors for Clostridium difficile-associated diarrhea (Kim
et al., 2010), an important disease with increasing rates of
mortality (Dawson et al., 2009). Likewise, a recent large
study involving 5387 elderly subjects, and a systematic
review of 2948 patients, have linked the use of PPIs with
an increased risk of diarrhea (Pilotto et al., 2008) and a
higher risk of enteric infections (Leonard et al., 2007),
respectively.
The mechanisms by which the suppression of gastric
acid secretion predisposes patients to an increased risk of
gastrointestinal (GI) disease are not well understood. For
example, while there is mounting evidence suggesting an
association between the use of PPIs and C. difficile-associ-
ated disease (Dial, 2009), gastric acid does not kill C. dif-
ficile spores (Rao et al., 2006), which are believed to be
crucial for the transfer of the microorganism (Dawson
et al., 2009). Also, a large case–control cohort study of
more than 170 000 ever-users of acid-suppressing drugs,
including PPIs, showed no association of antacid use with
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bacterial gastroenteritis (Garcia Rodriguez & Ruigomez,
1997) and a recent review of the literature indicates that
bacterial overgrowth during PPIs administration rarely
leads to clinical disease (Williams & McColl, 2006). These
observations illustrate the possibility that the development
of GI disorders in patients that are treated with gastric
acid inhibitors is a multi-factorial phenomenon rather
than an isolated association (Canani & Terrin, 2010).
The GI tract of mammals is home to a vast number of
different microbial groups, all acting in close symbiosis
with one another and with their host (Neish, 2009).
Despite the widespread medical use of PPIs and its poten-
tial involvement in intestinal dysbiosis (Vesper et al.,
2009), only a few studies have explored the effect of these
compounds on GI microbial communities, mainly using
culture techniques for specific microorganisms (e.g. Heli-
cobacter pylori) (Sharma et al., 1984; Fried et al., 1994;
Saltzman et al., 1994; Verdu et al., 1994; Logan et al.,
1995; Thorens et al., 1996). However, culture techniques
are by definition restricted to cultivable microorganisms,
a group representing a small proportion of all GI micro-
biota (Rajilic-Stojanovic et al., 2007). Culture indepen-
dent, 16S rRNA gene-based techniques have greatly
enhanced our knowledge of intestinal microbial inhabit-
ants, but these techniques have rarely been used to evalu-
ate the effect of gastric acid inhibition on the overall
composition of the GI bacterial microbiota (Williams &
McColl, 2006; Vesper et al., 2009).
As in humans, PPIs and other inhibitors of gastric acid
secretion are frequently used in dogs with disorders of
the upper GI tract. However, the effect of omeprazole or
any other suppressor of gastric acid secretion on the GI
bacterial microbiota of dogs has not been investigated in
detail and was the primary objective of this study.
Materials and methods
Study design
Eight intact clinically healthy mixed-breed dogs, four
male and four female, were entered into this study. All
dogs were 9 months old, of similar weight (18.6 ±2.0
kg), and were fed once a day with a commercial diet
(8755 Teklad: 21% protein, 4% fiber, Harlan). Omepra-
zole capsules (Zegerid, Santarus) were administered orally
at an average dose of 1.1 ±0.1 mg kg
1
(body weight)
twice a day (8 AM and 8 PM) for 15 days. Immediately
after administration of omeprazole, all dogs were given
20 mL of water orally. Multiple mucosal biopsy speci-
mens from the gastric body and the proximal duodenum
(12–15 from each site) were obtained from all dogs on
Days 30 (Day 30) and 15 (Day 15) before omeprazole
administration, on the last day of omeprazole administra-
tion (Day 15), and 15 days after the end of omeprazole
administration (Day 30). Biopsies were collected by
endoscopy under general anesthesia (sedation with but-
orphanol 0.2 mg kg
1
IM 15 min before induction with
thiopental IV 15 mg kg
1
followed by endotracheal intu-
bation and maintenance of anesthesia with sevoflurane in
100% oxygen via a circle system). For both stomach and
duodenum, three biopsies were flash frozen in liquid
nitrogen for DNA extraction and 6–7 biopsies were har-
vested and placed into 10% formalin for FISH analysis
and histological assessment according to the guidelines of
the World Small Animal Veterinary Association (Day
et al., 2008). Gastric juice (~5 mL) was obtained before
each endoscopic procedure via an endoscopic catheter,
and the pH measured immediately with a pH paper
(EMD Chemicals) and a pH meter. Fecal samples were
collected by rectal palpation on Days 30, 15, 15, and
30, and stored at 80 °C until analysis. The study proto-
col was approved by the Institutional Animal Care and
Use Committee of the University of Illinois (approval
number: 08261).
DNA extraction
Genomic DNA was extracted from the biopsies and feces
using a bead-beating phenol–chloroform method as
described elsewhere (Suchodolski et al., 2010).
Massive parallel 454-pyrosequencing
The gastric and duodenal mucosa-adherent microbiota
were evaluated using pyrosequencing of biopsy samples
collected on Days 30 and 15 (before omeprazole
administration), on Day 15 of omeprazole administration,
and on Day 30 (after discontinuation of omeprazole
administration) using a bacterial tag-encoded FLX-
Titanium 16S rRNA gene amplicon pyrosequencing (bTE-
FAP), as described previously for canine intestinal
samples (Handl et al., 2011). To estimate total bacterial
diversity, sequences were depleted of barcode primers,
chimeras, plastid, mitochondrial, and any non-16S bacte-
rial reads (<70% identity to any known high-quality 16S
sequence). Chimeras were depleted using Black Box Chi-
mera Check (B2C2) as described previously (Gontcharova
et al., 2010). Because sequence number may impact diver-
sity estimates, an equal number of high-quality sequences
were used for each sample. In order to use all available
samples, a total of 1000 sequences were selected randomly
from each sample, sequences <350 were removed and
the rest of the sequences trimmed to 350 bp and aligned
with MUSCLE (Edgar, 2004). The alignment was inspected
visually and deemed accurate. A distance matrix was cal-
culated from the alignment with PHYLIP (Felsenstein, 2005;
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gaps considered). Operational Taxonomic Units (OTUs)
then were assigned by MOTHUR using the read.otu com-
mand (Schloss et al., 2009).
To evaluate bacterial taxonomic community structure,
Phred20 quality reads were trimmed to remove tags and
primer sequences, depleted of chimera, plastid, mitochon-
drial, non-16S reads, and sequences <250 bp. The final
sequences were evaluated using BLASTN against a continu-
ally curated high-quality 16S rRNA gene database derived
from the National Center for Biotechnology Information
(NCBI) as described previously (Cephas et al., 2011).
BLAST outputs, based upon top-hit designations, were
compiled to generate percentage files at each taxonomic
level as described previously. Sequences with identity
scores to known or well-characterized 16S rRNA gene
sequences greater than 97% identity (<3% divergence)
were resolved at the species level, between 95% and 97%
at the genus level, between 90% and 95% at the family
level, between 85% and 90% at the order level, between
80% and 85% at the class level, and 77–80% at the phy-
lum level. Sequence information is available through Gen-
Bank within a short read archive (SRA) under accession
SRA045627.1.
Quantitative real-time PCR (qPCR)
In an effort to support the main findings of pyrosequenc-
ing (see below), the abundance of total bacteria, Helicob-
acter,Lactobacillus, and Enterococcus spp. was estimated
by qPCR in the obtained DNA samples from the gastric
and duodenal biopsies using published oligonucleotides
(Supporting Information, Table S1). TaqMan
®
reaction
mixtures (total 10 lL) contained 5 lL of TaqMan
®
Fast
Universal PCR master mix (29), No AmpErase
®
UNG
(Applied Biosystems), 1 lL of water, 0.4 lL of each pri-
mer (final concentration: 400 nM), 0.2 lL of the probe
(final concentration: 200 nM), 1 lL of 1% bovine serum
albumin (BSA, final concentration: 0.1%), and 2 lLof
DNA (1 : 10 or 1 : 100 dilution), and the PCR condi-
tions were: 95 °C for 20 s, and 40 cycles at 95 °C for 5 s,
and 10 s at the optimized annealing temperature (Table
S1). SYBR-based reaction mixtures (total 10 lL) con-
tained 5 lL of SsoFast
TM
EvaGreen
®
supermix (Biorad
Laboratories), 1.6 lL of water, 0.4 lL of each primer
(final concentration: 400 nM), 1 lL of 1% BSA (final
concentration: 0.1%), and 2 lL of DNA (1 : 10 or
1 : 100 dilution). PCR conditions were 95 °C for 2 min,
and 40 cycles at 95 °C 5 s and 10 s at the optimized
annealing temperature (Table S1). A melt curve analysis
was performed for SYBR-based qPCR assays under the
following conditions: 1 min at 95 °C, 1 min at 55°C, and
80 cycles of 0.5 °C increments (10 s each). Amplicons
were also visualized in an agarose gel (1%) to confirm
the presence of one band of the expected molecular size.
The qPCR data for Helicobacter,Lactobacillus, and Entero-
coccus spp. were normalized to the qPCR data for total
bacteria, and all samples were run in duplicate.
The abundance of total bacteria, Bifidobacterium,Lacto-
bacillus, the Bacteroides-Prevotella-Porphyromonas group,
gamma-Proteobacteria (Class), Firmicutes (Phylum), Clos-
tridium perfringens, as well as C. difficile and the C. diffi-
cile gene-encoding toxin B, was evaluated in feces using
published oligonucleotides (Table S1). The abundance of
Ruminococacceae (Family) and the genus Faecalibacterium
was also evaluated using family and genus-specific oligo-
nucleotides (as assessed by 16S rRNA gene clone libraries)
recently developed at our laboratory. The decision to tar-
get only a subset of fecal bacterial groups was based on
previous reports suggesting that these groups are highly
abundant phylotypes in feces of dogs (e.g. Ruminococca-
ceae) and because of purported beneficial properties (e.g.
Faecalibacterium and Bifidobacterium spp.). SYBR-based
qPCR assays were performed as described above (without
BSA) at the optimized annealing temperature (Table S1).
A commercial real-time PCR thermal cycler (CFX96TM;
Biorad Laboratories) was used for all qPCR assays. The
DNA concentration of all fecal samples was adjusted to
5nglL
1
.
Fluorescence in situ hybridization (FISH)
An average of six biopsies (range: 4–7 per organ evalu-
ated) were obtained at each time point and from each
dog, fixed in neutral-buffered 10% formalin for less than
48 h, and embedded in paraffin. Histological sections
(4 lm) were evaluated using FISH with oligonucleotide
probes 5′-labeled with 6-FAM or Cy-5 targeting the 16S
rRNA gene of total bacteria and Helicobacter spp. (Table
S1) as described previously (Jergens et al., 2009), with
minor modifications (no formamide in the hybridization
buffer in an effort to reduce toxic waste and because the
addition of formamide did not significantly increase the
fluorescent signal, and 48 and 50 °C for hybridization
and washing, respectively). Gastric and duodenal bacteria
were quantified every 3–5 microscopic fields throughout
the mucosal perimeter of each biopsy, depending on the
unique morphology of each specimen, using a Zeiss Stal-
lion digital confocal microscope (Carl Zeiss Microimag-
ing). To facilitate the quantification of bacteria at
different levels of the glass slide, at least three consecutive
pictures were taken sequentially throughout the vertical
z-axis (each picture separated from one another by
0.5 lm) from each microscopic field. A C-apochromat
(639water correction) objective lens was used for all
FISH analyses. Total bacteria were quantified using FISH
in both gastric (universal probe labeled with 6-FAM) and
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duodenal (universal probe labeled with Cy5) biopsies,
while the genus Helicobacter (FISH probes labeled with
Cy5) was only quantified in the gastric biopsies.
Statistical analysis
To assess the diversity of the GI microbiota, the Shannon
–Weaver (Shannon & Weaver, 1963) and Chao1 (Chao,
1987) diversity indices were calculated using MOTHUR.
Alterations of microbial communities before, during, and
after omeprazole administration were investigated using
an unweighted UniFrac distance matrix in the QIIME soft-
ware (Caporaso et al., 2010). In short, the high-quality
sequences as described above were aligned using MUSCLE,
and an optimized tree was generated (also using MUSCLE).
This tree served as the input tree for the unweighted
UniFrac distance metric.
Parametric analyses. A general linear mixed model
using the MIXED procedure of SAS
®
9.2 (SAS
®
Institute,
Inc.) was used to analyze the qPCR data with time,gen-
der, and time*gender interaction as fixed effects. The
inclusion of the interaction between time and gender is
justified by the fact that all dogs were the same age, had a
very similar body weight, and were subjected to the same
diet and environmental conditions. In addition, time was
also used in the REPEATED statement to model the
repeated measures (before, during, and after omeprazole
administration), and dog was included as a random effect.
The log
10
gastric Helicobacter FISH counts were analyzed
using a general linear mixed model in SAS
®
9.2 and the
same approach described for qPCR data. Post hoc multi-
ple comparisons were performed using the Tukey–Kramer
method. All model residuals showed a distribution very
close to normal, thus indicating valid models.
Nonparametric analyses. The Friedman’s test in Prism5
(GraphPad Software, CA) was used to compare the
pyrosequencing data (percentage of sequences) for each
bacterial group separately, gastric non-Helicobacter total
FISH counts, and the indexes of bacterial richness and
diversity. Post hoc multiple comparisons were performed
using the Dunn’s post-test. The NPAR1WAY procedure
in SAS
®
9.2 was used to compare intragastric pH and
duodenal bacterial FISH counts. A P-value of <0.05 was
considered to be statistically significant for all analyses.
Results
Side effects of omeprazole administration and
intragastric pH
All dogs remained clinically healthy throughout the study.
Intragastric pH was significantly increased during omep-
razole administration (median pH: 7.4, interquartile
range: 7.2–7.9) when compared with intragastric pH on
Days 30 (1.7, 1.5–1.9) and 15 (1.8, 1.5–2.1) before
administration, and Day 30 after omeprazole administra-
tion (1.5, 1.4–6.8) (P=0.0037). The pH measurements
did not correlate linearly or quadratically with gastric or
duodenal bacterial FISH counts, pyrosequencing, or qPCR
data (results not shown).
Number of sequences obtained by bTEFAP
A total of 142 026 (stomach) and 133 449 (duodenum)
sequences (~4000 per sample evaluated) were analyzed.
The number of sequences ranged from 1199 to 7734
sequences in gastric samples and 1028–9383 sequences
in duodenal samples. On average, the number of
sequences per dog varied from 3176 to 5476 in gastric
samples and from 1955 to 6590 in duodenal samples.
With the exception of the gastric microbiota of two
male dogs at only one different time point each, the
gastric and duodenal microbiota formed completely sep-
arated phylogenetic clusters (Fig. S1), suggesting a dis-
tinctive microbiota in each of the evaluated segments of
the GI tract.
bTEFAP in gastric biopsies
In the stomach, a median of 36 OTUs (>97%
sequence identity, range: 18–119) was detected per dog
per time point. There was a higher bacterial richness in
the stomach during omeprazole administration (median:
56, range: 26–70; median before and after: 35, range:
18–119) but this difference did not reach significance
(Table 1). Bacterial diversity was not significantly modi-
fied. While we observed significant changes in specific
bacterial groups in response to omeprazole administra-
tion (see below), the constructed dendrograms based
on the Unifrac distance metric did not reveal an obvi-
ous clustering of animals according to treatment period
(Fig. S2). The great majority (>90% on average at
baseline) of the obtained sequences from the stomach
were classified as Proteobacteria, a phylum that
decreased in its relative abundance during omeprazole
administration (P=0.0427, Table S2). This effect was
more evident on the genus Helicobacter (P=0.0336).
The median percentage of Helicobacter spp. sequences
during omeprazole administration was 92%, while the
median percentage before and after omeprazole was
>98%. This decrease in the relative abundance of
Helicobacter spp. during omeprazole administration was
accompanied by an increase in the relative abundance
of other genera of the phyla Proteobacteria (especially
Actinobacillus), Firmicutes (especially Streptococcus), and
Fusobacteria (Table S2).
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FISH in gastric biopsies
Gastric Helicobacter spp. and non-Helicobacter bacteria
were counted throughout the mucosal side of a total
of 155 gastric biopsies from a similar number of
microscopic fields (Table S3 and Fig. S3). There was a
significant effect of omeprazole on the abundance of
gastric Helicobacter (P<0.0001), and there was no
difference in abundance of gastric Helicobacter between
the male and female dogs (P=0.3161) (Fig. 1). Also,
there was a significant interaction between time and
gender (P=0.0323), suggesting that the change in
gastric Helicobacter organisms over time was different
between the male and female dogs (Fig. 1). Also, in
the stomach, non-Helicobacter bacteria were observed
more frequently during omeprazole administration
(median: 3, range: 0–20) than on Day 30 (median:
0, range: 0–3) and Day 15 (median: 1, range: 0–6)
before omeprazole administration, and 15 days after
omeprazole administration on Day 30 (median: 0,
range: 0–2) (P=0.0300).
Quantitative real-time PCR in gastric biopsies
There was no significant effect of omeprazole administra-
tion on gastric total bacteria (P=0.0687), there was no
difference in bacterial abundance between the male and
female dogs (P=0.7566), but there was a significant
interaction between time and gender (P<0.0001)
(Fig. 2). In the male dogs, there was a higher bacterial
abundance during omeprazole administration on Day 15
(P=0.0093) and on Day 30 after discontinuation of
omeprazole administration (P=0.0007) when compared
with that on Day 30 before omeprazole administration
(Fig. 2). There was no significant effect of omeprazole
administration on total gastric bacteria in the female
dogs, and there was no significant effect of omeprazole
administration on the abundance of gastric Helicobacter
and Lactobacillus spp. (Fig. 2).
bTEFAP in duodenal biopsies
In the duodenum, a median of 173 OTUs (>97%
sequence identity, range: 52–285) was detected per dog
per time point. Omeprazole administration was not asso-
ciated with significant differences in the indexes of bacte-
rial richness and/or diversity (Table 1). While we
observed significant changes in specific bacterial groups
in response to omeprazole administration (see below), the
constructed dendrograms based on the Unifrac distance
metric did not reveal any obvious clustering of animals
according to treatment period (Fig. S4). Bacterial
Table 1. Median (interquartile range) indices of bacterial diversity (Shannon–Weaver and Chao1 3%) and richness (OTU 3%) on Days 30 and
15 before omeprazole administration, during omeprazole administration (Day 15), and after discontinuation of omeprazole administration (Day
30). P-values were obtained by nonparametric Friedman’s tests.
Day 30 Day 15 Day 15 Day 30 P-values
Stomach
Shannon 1.2 (0.9/1.6) 1.4 (1.0/1.9) 1.4 (0.9/2.4) 0.9 (0.8/1.3) 0.3476
Chao1 59 (38/81) 77 (48/83) 98 (56/194) 47 (38/74) 0.0917
OTU 35 (29/40) 37 (31/45) 56 (38/92) 30 (24/49) 0.2468
Duodenum
Shannon 4.1 (3.4/4.6) 3.9 (3.6/4.3) 2.9 (2.4/4.2) 3.3 (2.7/4.0) 0.1447
Chao1 324 (274/470) 387 (229/453) 236 (131/395) 239 (142/471) 0.1116
OTU 184 (140/246) 191 (129/228) 112 (85/218) 131 (103/220) 0.2898
These estimates are based on 1000-sequence subsamples. Chao1 and OTU estimates were rounded up to better fit in the table.
Fig. 1. Log
10
Helicobacter FISH counts per microscopic field on Day
30 (D 30) and Day 15 (D 15) before omeprazole administration,
the last day of omeprazole administration (D 15), and 15 days after
the completion of omeprazole administration (D 30) in the male (left)
and the female (right) dogs. The error bars represent the mean and
the standard error. Within each gender, there was a significant
decrease in gastric Helicobacter spp. during omeprazole
administration at Day 15 (*D15) when compared to all other time
points (P<0.0001).
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representatives of at least seven different phyla were iden-
tified in the duodenum (Table S4). The majority of the
obtained sequences from the proximal duodenum were
classified as Firmicutes, followed by Proteobacteria and
Bacteroidetes. On average, these three bacterial phyla com-
prised more than 80% of all sequences at all time points.
Omeprazole administration was associated with a higher
relative abundance of Enterococcus (P=0.0137) and a
lower relative abundance of Helicobacter (P=0.0287) and
Porphyromonas (P=0.0316), but there was no statistically
significant difference in all the rest of the bacterial groups
analyzed (Table S4).
Interestingly, all four male dogs had an increase in the
Class Bacilli (Phylum Firmicutes) during omeprazole
administration (all had >70% during omeprazole admin-
istration, while only two had more than 10% at either
baseline evaluation) (Fig. S5). This effect was also evident
at the order Lactobacillales and the genera Enterococcus
and Lactobacillus in three of the four male dogs. This
consistent increase in Bacilli during omeprazole adminis-
tration in the male dogs was associated with a lower
abundance of other bacterial phyla (especially Proteobacte-
ria and Bacteroidetes) during omeprazole administration.
In the female dogs, no such consistent changes in the
proportions of duodenal bacteria were observed.
FISH in duodenal biopsies
Duodenal total bacteria were counted in a total of 132
biopsies from a similar number of microscopic fields
(Table S3). While the median number of bacteria per
microscopic field was zero for all time points (range: 0–3),
nonparametric analyses revealed higher numbers of bacte-
ria during omeprazole administration (P=0.0033). The
sum of all counted bacteria during omeprazole adminis-
tration was 40 bacteria (male dogs only: 34), while
the median sum of all other time points was eight bacte-
ria. All the observed bacteria were morphologically similar
Fig. 2. Quantitative real-time PCR results for total gastric bacteria (a), gastric Helicobacter (b), and gastric Lactobacillus spp. (c) on Day 30 (D
30) and Day 15 (D 15) before omeprazole administration, the last day of omeprazole administration (D 15), and 15 days after the completion
of omeprazole administration (D 30) in the male (left) and the female (right) dogs. Error bars represent the mean and the standard error.
Horizontal brackets represent statistical significance (P<0.01). qPCR data for Helicobacter and Lactobacillus spp. were normalized to qPCR data
for total bacteria.
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(i.e. rod-shaped, 2–3lm long). It is possible that we
underestimated bacterial populations using FISH owing
to issues with probe penetration and/or over-fixation with
formalin.
Quantitative real-time PCR in duodenal
biopsies
There was a significant effect of omeprazole administra-
tion on the abundance of total duodenal bacteria
(P=0.0003), but there was no difference between gen-
ders and there was no significant interaction between
omeprazole administration and gender. Regardless of gen-
der, there was a higher bacterial abundance on Day 15
during omeprazole administration when compared to
Day 15 before omeprazole administration (P=0.0295).
Also, there was a higher bacterial abundance in the duo-
denum on Day 30 after omeprazole administration when
compared to that on Day 30 (P=0.0040) and Day 15
(P=0.0009) before omeprazole administration (Fig. 3).
In contrast to the pyrosequencing results that showed a
decrease of Helicobacter spp. in the duodenum during
omeprazole administration, the genus Helicobacter was
detected only at six isolated time points in the duodenum
of five dogs (three male and two female dogs). Enterococ-
cus spp. was detected only in two male dogs during
omeprazole administration on Day 15. There was a signif-
icant effect of omeprazole on duodenal Lactobacillus spp.
(P<0.0001) with male dogs having a higher abundance
of duodenal Lactobacillus when compared with female
dogs (P=0.0168). Also, there was a significant interac-
tion between omeprazole administration and gender
(P<0.0001) (Fig. 3). The male dogs had a significantly
higher abundance of Lactobacillus spp. in the duodenum
during omeprazole administration when compared to all
time points before and after omeprazole administration
(P<0.0001 for all multiple comparisons) (Fig. 3).
Quantitative real-time PCR for the analysis of
fecal microbiota
One fecal DNA sample (from one female dog, Day 15
before omeprazole administration) was not available and
was treated as a missing value. All time points in all dogs
were PCR negative for C. difficile and the C. difficile
gene-encoding toxin B. C. perfringens was detected in all
female dogs on Day 30 before omeprazole administra-
tion only. Regardless of gender, there was a significant
increase in fecal Lactobacillus during omeprazole adminis-
tration when compared with all other time points
(P<0.0001, Fig. 4). This increase in Lactobacillus was
accompanied, in the male dogs only, by a decrease of
Faecalibacterium and the Bacteroides-Prevotella-Porphyro-
monas group (Fig. 4).
Fig. 3. Quantitative real-time PCR results for total duodenal bacteria (a) and Lactobacillus spp. (b) on Day 30 (D 30) and Day 15 (D 15)
before omeprazole administration, the last day of omeprazole administration (D 15), and 15 days after the completion of omeprazole
administration (D 30). Error bars represent the mean and the standard error. Horizontal brackets represent statistical significance (P<0.0001).
*Significantly different (P=0.0295) than Day 15 before omeprazole administration (D 15), regardless of gender. †Significantly different than
Day 15 (P=0.0040) and Day 30 (P=0.0009) before omeprazole administration, regardless of gender. qPCR data for Lactobacillus spp. were
normalized to qPCR data for total bacteria.
ª2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 80 (2012) 624–636
Published by Blackwell Publishing Ltd. All rights reserved
630 J.F. Garcia-Mazcorro et al.
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Discussion
PPIs and other suppressors of gastric acid secretion are
used extensively in both human and veterinary patients
with suspected disorders of the upper GI tract. Despite
the widespread use of these compounds in dogs and
the cumulative evidence suggesting an association
between PPI use and GI infections in human patients,
there are no studies to date that have evaluated the
effect of PPIs or any other gastric acid suppressor on
the composition of the canine GI microbiota. The
results of this study suggest that orally administered
omeprazole at a dose of 1.1 mg kg
1
twice a day for
15 days can alter the quantitative composition of the
gastric, duodenal, and fecal bacterial microbiota of
healthy dogs.
In this study, omeprazole administration led to a
decrease in gastric Helicobacter spp., an effect which was
more evident on the quantitative FISH analysis. While a
growing number of investigations suggest that PPIs can
also lead to a decrease in the abundance of gastric
H. pylori in humans, most studies have evaluated the effect
of PPIs on this bacterium only in combination with other
pharmaceuticals such as antibiotics (Graham & Fischbach,
2010; Luther et al., 2010; Wu et al., 2010). Also, the histo-
logical density of H. pylori in the gastric body and antrum
of humans was reduced after 4 weeks of omeprazole treat-
ment, while it was increased in the fundus (Logan et al.,
Fig. 4. Quantitative real-time PCR results for all fecal bacteria (a), Firmicutes (b), Ruminococcaceae (c), Faecalibacterium (d), Lactobacillus (e), the
Bacteroides-Prevotella-Porphyromonas group (f), Gammaproteobacteria (g), and Bifidobacterium (h) on Days 30 (Day 30) and 15 (Day 15)
before omeprazole administration, during omeprazole administration on Day 15, and 15 days after the completion of omeprazole administration
(Day 30). Error bars represent the mean and the standard error. Horizontal brackets represent statistical significance (P<0.01). There was a
significant interaction (P<0.05) between omeprazole administration and gender for all fecal bacteria (a), Faecalibacterium (d), the Bacteroides-
Prevotella-Porphyromonas group (f), and Gammaproteobacteria (g). *Statistically significantly different (P<0.0001) than all other time points
regardless of gender. Notice that the y-axis is in a different scale for each bacterial group.
FEMS Microbiol Ecol 80 (2012) 624–636 ª2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Effect of omeprazole on gastrointestinal microbiota of dogs 631
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1995). Other studies have confirmed this phenomenon
(Ishihara et al., 2001). In the current study, we collected
biopsies only from the gastric body and antrum, and
therefore, we cannot confirm an overall decrease in gastric
Helicobacter in all regions of the stomach. Moreover, the
quantitative real-time PCR assay used in this study did
not confirm the decrease in gastric Helicobacter spp. abun-
dance during omeprazole administration, an effect sug-
gested by both bTEFAP and FISH. It is possible that the
qPCR assay used here does not detect all canine gastric
species and strains of Helicobacter. For instance, while
both the reverse primer and the oligo probe detect all
Helicobacter spp. that have been isolated from the stomach
of dogs (Neiger & Simpson, 2000), the forward primer
may not detect H. bilis and Flexispira rappini (see Table
S1). The latter may be especially relevant as it includes
multiple Helicobacter taxa (Dewhirst et al., 2000). These
observations raise the interesting question of whether the
effect of omeprazole is different among different species
and/or strains of gastric Helicobacter, a hypothesis that is
indirectly supported by a recent study showing that the
effect of pantoprazole (another PPI) on growth and mor-
phology of bacteria was different among several strains of
oral Lactobacillus spp. (Altman et al., 2008).
The mechanism by which omeprazole leads to a
decrease in gastric Helicobacter is unclear and controver-
sial (Canani & Terrin, 2010). Omeprazole could have an
indirect effect by means of raising intragastric pH, which
in turn could allow other non-Helicobacter bacteria to
thrive. Alternatively, omeprazole may act directly by
means of a direct bactericidal effect. For instance, it has
been shown that omeprazole inhibits the growth of gram-
positive and gram-negative bacteria in vitro, including
H. pylori (Jonkers et al., 1996). More recent studies also
support a direct effect of PPIs on H. pylori (Suzuki et al.,
2003; Nakamura et al., 2007). This effect may be due to a
direct effect on the proton pumps of the bacteria, as these
enzymes have been identified at least in H. pylori (Mel-
chers et al., 1998) and Streptococcus pneumoniae (Hoskins
et al., 2001). Thus, it has been hypothesized that these
enzymes of bacterial origin may serve as extrinsic sites of
action for PPI therapy (Vesper et al., 2009). However,
while much research has focused on H. pylori, dogs are
not known to harbor this species in the stomach but
other Helicobacter spp. such as H. felis and H. heilmannii
(Neiger & Simpson, 2000; Shinozaki et al., 2002). To
date, the effect of PPIs on other non-H. pylori gastric
Helicobacter spp. has not been investigated.
The decrease in gastric Helicobacter abundance during
omeprazole administration was accompanied by a higher
relative abundance of other bacteria, especially Streptococ-
cus,Lactobacillus, Fusobacterium, and Actinobacillus,
as suggested by pyrosequencing. It is likely that other,
non-Helicobacter bacteria were able to thrive in the stom-
ach during the temporary reduction in intragastric acid-
ity. It is also possible that some of these bacteria possess
a direct antagonist effect against Helicobacter spp., as sug-
gested by a recent study of the effect of two strains of
Lactobacillus on H. pylori (Cui et al., 2010). However, it
is not clear whether the bacteria that were found more
abundantly during omeprazole administration were native
to the stomach or foreign, for example, from the mouth
and esophagus. One study suggested that the human
stomach could contain its own distinct microbial ecosys-
tem (Bik et al., 2006), but the authors warned that this
observation was based on a comparison of gastric, oral,
and esophageal bacterial communities from different sub-
jects with different clinical syndromes.
In the duodenum, omeprazole led to an increased rela-
tive abundance in Lactobacillus and Enterococcus in the
male dogs, which likely caused the observed higher abun-
dance of all bacteria suggested by FISH analysis. In the
past, an abnormal accumulation of bacteria in the small
bowel of dogs was termed as small intestinal bacterial
overgrowth (SIBO; Johnston, 1999), but the understand-
ing of this phenomenon has undergone several advances
(Hall, 2011), in part because of the complex microbial
composition discovered in the canine small intestine
(Mentula et al., 2005; Suchodolski et al., 2008a, b; Xe-
noulis et al., 2008; Suchodolski et al., 2009, 2010). In
small animal veterinary medicine, small intestinal dysbio-
sis is a currently used term to define a clinical syndrome
caused by an alteration, either qualitative, quantitative, or
both, of one or more groups of the small intestinal mic-
robiota. Although the observed changes in the composi-
tion of the duodenal microbiota during omeprazole
administration may be considered a dysbiosis (from its
baseline composition), its clinical significance remains to
be determined.
In addition to the changes in the stomach and duode-
num, our results also suggest that omeprazole can alter
the composition of the fecal microbiota. Similarly, one
recent study showed that orally administered omeprazole
can lead to changes in fecal microbial communities of
mice in a dose-dependent manner (Kanno et al., 2009).
However, unlike the current study that showed a higher
abundance of some fecal bacteria (e.g. Lactobacillus)
accompanied by a lower abundance of other bacteria (e.g.
Faecalibacterium and Bacteroides) during omeprazole
administration, Kanno et al. showed that all groups of
fecal bacteria (with the exception of Bifidobacterium)
increased during omeprazole administration in mice
(Kanno et al., 2009). Because omeprazole is metabolized
by the hepatic cytochrome P450 system after absorption
from the small intestine and about 80% of the metabo-
lites are excreted in urine (Petersen, 1995), it is unlikely
ª2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 80 (2012) 624–636
Published by Blackwell Publishing Ltd. All rights reserved
632 J.F. Garcia-Mazcorro et al.
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that omeprazole reaches the large intestine, at least in its
native form. Thus, our results and the results reported by
Kanno et al. suggest that it is the increase in the bacterial
load entering the large intestine that is responsible for the
changes observed in the fecal microbiota. Another factor
affecting the fecal microbiota during inhibition of gastric
acid could be the change in the composition of dietary
protein reaching the large intestine (Zentek et al., 2003),
as gastric acid plays a key role in the initial stages of pro-
tein digestion. It seems likely that both mechanisms con-
tribute to the changes observed in the fecal microbiota.
The decrease in Faecalibacterium during omeprazole
administration in the male dogs is especially interesting,
as these bacteria possess anti-inflammatory properties
(Sokol et al., 2008) and have been found to be depleted
during episodes of colitis in humans (Sokol et al., 2009).
Finally, the interaction between the effect of omepra-
zole on the GI bacterial microbiota and gender suggested
in this study may deserve scrutiny in future studies. Inter-
estingly, Zhang et al. (2006) showed that higher endoge-
nous progesterone concentrations in women could have a
stimulatory effect on the P450 3A (CYP3A) activity,
which plays an essential role in the metabolism of omep-
razole in the liver (Andersson et al., 1993, 1994). How-
ever, all the females in the current study did not show
signs of their first heat season until weeks after the last
sample collection, and it has been shown that bitches
have undetectable serum concentrations of progesterone
during anestrous (Hase et al., 1999).
In summary, this study suggests that orally adminis-
tered omeprazole can alter the quantitative abundance of
several bacterial communities throughout the GI tract of
healthy dogs. Particularly, in this study, omeprazole
administration was associated with a decrease in Helicob-
acter spp. and an increase of other bacteria in the stom-
ach. Also, omeprazole administration was associated with
higher numbers of total bacteria and an increase in Lacto-
bacillus in the duodenum of the male dogs. Lastly, omep-
razole led to an increase in fecal Lactobacillus, which was
accompanied by a decrease in Faecalibacterium and the
Bacteroides-Prevotella-Porphyromonas group in the male
dogs. However, omeprazole administration was not asso-
ciated with major qualitative changes in the phylogenetic
composition of the stomach and the duodenum, as evalu-
ated by Unifrac analysis of pyrosequencing results. Fur-
ther studies are warranted to investigate the clinical
significance of these findings.
Acknowledgements
Part of this investigation was presented in the form of an
abstract in the Forum 2010 and 2011 of the American
College of Veterinary Internal Medicine. The study was
funded by the College of Veterinary Medicine at the Uni-
versity of Illinois and the Gastrointestinal Laboratory at
Texas A & M University.
The authors acknowledge Angie Otto and Megan Miller
for their help during the animal phase of the study.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Dendrogram illustrating the phylogenetic cluster-
ing of the gastric (GB: gastric biopsy) and duodenal bac-
terial microbiota.
Fig. S2. Dendrogram illustrating the phylogenetic cluster-
ing of the gastric microbiota.
Fig. S3. Serial images of gastric Helicobacter spp. through-
out the vertical z-axis in one microscopic field from a
gastric biopsy.
Fig. S4. Dendrogram illustrating the phylogenetic cluster-
ing of the duodenal microbiota.
Fig. S5. Percentage of sequences in the male (left) and
the female (right) dogs at the class Bacilli (a), order Lac-
tobacillales (b), and Lactobacillus on Day 30 (Day 30)
and Day 15 (Day 15) before omeprazole administration,
the last day of omeprazole administration (Day 15), and
on Day 30 after discontinuation of omeprazole adminis-
tration.
Table S1. Oligonucleotides used in this study for quanti-
tative real-time PCR (qPCR) assays and fluorescent in situ
hybridization (FISH).
Table S2. Median (interquartile range) proportions of py-
rosequencing tags in the stomach on Day 30 (Day 30)
and Day 15 (Day 15) before omeprazole administration,
the last day of omeprazole treatment (Day 15), and 15
days after omeprazole treatment (Day 30).
Table S3. Number of microscopic fields analyzed for each
gender for FISH analyzes in the gastric (stomach) and the
duodenal (duodenum) biopsies.
FEMS Microbiol Ecol 80 (2012) 624–636 ª2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
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Table S4. Median (interquartile range) proportions of
pyrosequencing tags in the duodenum on Day 30 (Day
30) and Day 15 (Day 15) before omeprazole adminis-
tration, the last day of omeprazole treatment (Day 15),
and 15 days after omeprazole treatment (Day 30).
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material) should be directed to the corresponding author
for the article.
ª2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 80 (2012) 624–636
Published by Blackwell Publishing Ltd. All rights reserved
636 J.F. Garcia-Mazcorro et al.
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