- Email: [email protected]
The effect of penicillin administration in early life on murine gut microbiota and blood lymphocyte subsets
Accepted Manuscript The effect of penicillin administration in early life on murine gut microbiota and blood lymphocyte subsets Jaroslaw Daniluk, Urszula Daniluk, Malgorzata Rusak, Milena Dabrowska, Joanna Reszec, Magdalena Garbowicz, Kinga Humińska, Andrzej Dabrowski PII:
S1075-9964(17)30058-6
DOI:
10.1016/j.anaerobe.2017.03.015
Reference:
YANAE 1711
To appear in:
Anaerobe
Received Date: 24 December 2016 Revised Date:
4 March 2017
Accepted Date: 15 March 2017
Please cite this article as: Daniluk J, Daniluk U, Rusak M, Dabrowska M, Reszec J, Garbowicz M, Humińska K, Dabrowski A, The effect of penicillin administration in early life on murine gut microbiota and blood lymphocyte subsets, Anaerobe (2017), doi: 10.1016/j.anaerobe.2017.03.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The effect of penicillin administration in early life on murine gut microbiota
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and blood lymphocyte subsets.
3 Jaroslaw Daniluk1, Urszula Daniluk2, Malgorzata Rusak3, Milena Dabrowska3,
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Joanna Reszec4, Magdalena Garbowicz5, Kinga Humińska5, Andrzej Dabrowski1
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Bialystok ul. M. Sklodowskiej-Curie 24a, 15-276 Bialystok, Poland
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Department of Gastroenterology and Internal Medicine, Medical University of
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Department of Pediatrics, Gastroenterology and Allergology, Medical University of
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Bialystok ul. J. Waszyngtona 17, 15-274 Bialystok, Poland
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Waszyngtona 15A, 15-269 Bialystok, Poland
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Waszyngtona 13, 15-269 Bialystok, Poland
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Poland
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Department of Medical Pathomorphology, Medical University of Bialystok ul. J.
Genomic Laboratory, DNA Research Center ul. Mickiewicza 31, 60-385 Poznan,
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Department of Haematological Diagnostics, Medical University of Bialystok ul. J.
Corresponding author: Jaroslaw Daniluk, Department of Gastroenterology and
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Internal Medicine, Medical University of Bialystok ul. M. Sklodowskiej-Curie 24a, 15-
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276 Bialystok, Poland
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Phone: 48 85 746 82 34
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fax: 48 85 746 85 06
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Email: [email protected], [email protected]
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Abstract Background and aim: Antibiotics have many beneficial effects but their
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uncontrolled use may lead to increased risk of serious diseases in the future. Our
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hypothesis is that an early antibiotic exposition may affect immune system by
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altering gut microbiota. Therefore, the aim of the study was to determine the effect of
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penicillin treatment on gut microorganisms and immune system of mice. Methods:
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21-days old C57BL6/J/cmdb male mice were treated with low-dose of penicillin
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(study group) or water only (control group) for 4 weeks. Tissue and stool samples for
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histology or microbiome assessment and peripheral blood for CBC and flow
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cytometry evaluation were collected. Results: We found high variability in microbiota
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composition at different taxonomic levels between littermate mice kept in the same
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conditions, independently of treatment regimen. Interestingly, low-dose of penicillin
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caused significant increase of Parabacteroides goldsteinii in stool and in colon tissue
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in comparison to control group (9.5% vs. 4.9%, p=0.008 and 10.7% vs. 6.1%,
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p=0.008, respectively). Moreover, mice treated with penicillin demonstrated
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significantly elevated percentage of B cells (median 10.5% vs 8.0%, p=0.01) and
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decrease in the percentage of total CD4+ cell (median 75.4% vs 82.5%, p=0.0039)
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with subsequent changes among subsets - increased percentage of regulatory T
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cells (Treg), T helper 1 (Th1) and T helper 2 (Th2) cells. Conclusion: Our study
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showed significant effect of penicillin on B and T cells in peripheral blood of young
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mice. This effect may be mediated through changes in gut microbiota represented
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by the expansion of Parabacteroides goldsteinii.
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Key words: lymphocytes, microbiota, Parabacteroides goldsteinii, penicillin
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1. Introduction Discovery of penicillin in 1928 by Sir Alexander Fleming, saved many lives
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and revolutionized treatment of bacterial infections. Antibiotics have also found
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application in agriculture, due to their effect on weight gain (up to 15%) in farm
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animals [1]. Nowadays easy access to the antibiotics led to their overuse in humans
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and caused development of bacterial resistance to the treatment.
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Despite of their benefit in infection treatment, antibiotics have profound
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influence on the structure and function of gut microbes. Our knowledge about human
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microbiome has dramatically increased over the last decade due to development of
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novel sequence-based molecular tests, like 16s rRNA-based sequencing, which
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became the gold standard technique in microbiome identification [2]. Commensal
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microorganisms prevent colonization of the gut by pathogens. They are involved in
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the process of appropriate development and maturation of innate and acquired
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immune system during the childhood, and maintaining homeostasis in the adults [3,
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4]. The fetal gastrointestinal tract is sterile but immediately after the birth it is
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colonized by many different microorganisms [5]. Recently, it has been reported that
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any perturbations in microorganisms composition caused by antibiotics or changes
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in the diet habits during the first two years of live, may predispose to the
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development of chronic diseases like obesity, type 1 diabetes, rheumatoid arthritis,
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coeliac disease, liver diseases and inflammatory bowel disease (IBD) [6-8]. It has
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been also suggested that antibiotic exposure during the first year of life increases
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the risk of asthma development [9]. Jernberg et al reported that even a short, 1-week
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long course of antibiotic regimen causes significant and prolonged (up to 2 years)
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changes in microbiota composition [10]. Therefore, the aim of our study was to
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determine the effect of early antibiotic (penicillin) exposition on gut microorganisms
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and immune system of mice.
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74 2. Material and methods 2.1. Animals
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C57BL6/J/cmdb male mice (Medical University of Bialystok; Bialystok, Poland) were
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maintained in specific pathogen-free (SPF) conditions. All in vivo experiments were
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performed according to EU Directive 2010/63/EU and approved by the Local
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Committee for Experiments with the Use of Laboratory Animals, Bialystok, Poland.
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21-days old mice, just after the weaning, were randomly divided into two groups:
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study group – animals receiving penicillin dissolved in drinking water (n=10) and
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control group – mice receiving drinking water only (n=10), ad libitum for 28 days.
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Dose of the antibiotic, 1 µg of penicillin / 1 gram of body weight, was determined on
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the basis of previous literature data and assumption that daily consumption of water
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is 15 ml per 100 g of mice [11, 12]. Fresh containers with water and water with
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antibiotic were changed three times a week. Mice were housed 5 per cage and they
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were allowed to eat standard chow diet ad libitum. Consumption of water or water
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with penicillin was determined on daily basis for each cage. Body weight of each
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mouse was measured every fourth day.
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2.2. Sample Collection
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After 28 days, mice were anesthetized and sacrificed. Blood samples were collected
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by cardiac puncture and divided into two parts - complete blood count (CBC) and
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flow cytometry analysis. The small and large intestine, pancreas, and liver were
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harvested aseptically and stored for further histological evaluation. For microbiota
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assessment samples were collected directly from tissue (distal part of small intestine
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and ascending colon), and from the stool (lumen of the caecum), and immediately
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snap-frozen and stored (-80°C).
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2.3. CBC and flow cytometry analysis of isolated cells
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To determine CBC, 200 µl of peripheral blood was used and analyzed in the
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Department of Haematological Diagnostics, Medical University of Bialystok.
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Peripheral subpopulations of lymphocytes were assessed using flow cytometry. To
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prepare samples, approximately 200 µl of peripheral blood was treated with RBC
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lysis buffer (Sigma-Aldrich) for 10 min at room temperature, and the remaining cells
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were washed twice with cold PBS and centrifuged at 1,200 rpm for 10 min. Cells
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were stained with the appropriate combinations of the following antibodies: FITC–
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anti-CD3e (145-2C11; BD Pharmigen), APC-anti-CD4 (MR4-5; BD Pharmigen), PE-
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anti-CD25 (3C7; BD Pharmigen), PE-Cy7-anti-CD127 (SB/199; BD Pharmigen), and
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Mouse T Lymphocyte Subset Antibody Cocktail with Isotype Control (BD Pharmigen)
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containing PE-Cy 7- anti-CD3e (145-2C11), PE-anti-CD4 (RM4-5), APC-anti- CD8a
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(53-6.7), Mouse B Lymphocyte Subset Antibody Cocktail with Isotype Control (BD
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Pharmigen) with PE-Cy 7- anti-CD45R/B220 (145-2C11); PE-anti-CD23 (RM4-5);
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APC-anti- sIgM (53-6.7). The Mouse Th1/Th2/Th17 Phenotyping Kit (PerCP-Cy5.5-
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anti-CD4 (RM4-5); PE-anti-IL-17A (TC11-18H10.1); FITC-anti-INF-GMA (XMG1.2);
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APC-anti-CD4 (11B11) BD Pharmigen) were used according to the manufacturer’s
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instruction for the detection of CD4+IL-17+, CD4+IFNγ+, CD4+IL-4+ expression.
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Flow cytometric data were acquired using a FACS Canto II cytometer with BD
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FACSDiva Software v6.1.3 (BD Biosciences) and analyzed with Worksheet software
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(BD).
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2.4. Histology and Immunohistochemistry
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The organs were fixed with 10 % PBS-buffered formalin for 24h, embedded in
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paraffin, cut sagittally into 5-µm sections, stained with hematoxylin and eosin (H&E),
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and examined by light microscopy (Olympus BX45) for histological analysis. For
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each animal, ten fields at a magnification of ×100 were captured randomly from the
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four different parts of the intestine.
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To determinate the lymphocytic infiltration we used antibodies against T cytotoxic
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cells, T helpers and against lymphocytes B. Following the deparaffinization and
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rehydration, epitope retrieval was carried out in the EnVision Flex Target Retrieval
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Solution (DAKO) at low pH. Endogenous peroxidases were blocked by incubating
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the sections in methanol and 3% hydrogen peroxidase for 40 minutes. Next, slides
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were incubated with proper anti-mouse antibodies against CD3, CD4, CD8 and
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CD20 in 1:100 dilutions for 1 hour at room temperature. Visualization reagent
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EnVision (DAKO) was applied for 30 minutes and followed by DAB solution for 10
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minutes. The slides were than counterstained with H&E and examined under the
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light microscope. The intensity of immunostaining was evaluated in random 10 fields
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under ×20 magnification. Appropriate positive and negative controls were performed.
140 2.5. Faecal microbiota analysis
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The bacterial composition of small and large intestine, and stool samples of each
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group was carried out using 16S ribosomal RNA (rRNA) gene sequencing on the
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MiSeq apparatus (Illumina) via next-generation sequencing (NGS) technology. In
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order to analyze bacterial components, the V3-V4 hypervariable region of the 16S
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rRNA gene was firstly amplified from the genomic DNA extracted from fecal samples
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by
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GACTACHVGGGTATCTAATCC-3’)
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sequences, compatible with Illumina’s indices. Incorporation of primers with indexes
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and adapters was followed by second PCR reactions. After each amplification step,
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the PCR products were purified by using AMPure XP beads (Beckman Coulter
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Genomic, CA, USA). DNA amplified fragments were normalized to 2nM by Qubit®
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dsDNA HS kit (high
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sequencing. 8pM libraries were loaded on the MiSeq plarform (Illumina Inc., San
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Diego, CA, USA). Sequencing was performed with 300-nucleotide-long paired-end
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reads on the Illumina sequencer (MiSeq) according to the instructions of the
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manufacturer. The analysis output file generated for the 16S metagenomics
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workflow provided classification of reads for each sample. Obtained data were
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analyzed and interpreted using specialized software packages: Quantitative Insights
primers:
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(5’-CCTACGGGNGGCWGCAG-3’) containing
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into Microbial Ecology (QIIME), Greengenes, Metagenome Analyzer (MEGAN) I
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Ribosomal Database Project (RDP).
162 2.6. Data analysis
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Data were analyzed using Statistica 10 software. Statistical significance was
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determined by U Mann-Whitney test; p<0.05 was considered statistically significant.
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3.1. Body weight, CBC and histological evaluation
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During the whole study period, body weights of animals were within the range of
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growth for C57BL6/J/cmdb male mice. There was no statistically significant
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difference in body weight change between control and study groups (Table 1). There
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was no difference in consumption of water in control (mean 21.8 ml/cage/day) or
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water with penicillin in study group (mean 23.0 ml/cage/day). After 28 days of
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penicillin administration no differences in white blood count, red blood count or
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platelets count were observed between the groups. However, 4 weeks of antibiotic
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exposure significantly increased percentage of blood neutrophils in comparison to
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control group (6.0% vs. 4.0%, p=0.0002). This was accompanied by significant
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decrease of lymphocytes percentage in the treatment group in comparison to control
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(92.5% vs. 95.0% respectively, p=0.0002).
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Histological examination of the small and large intestinal biopsies revealed no
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abnormalities between study groups (Fig. 1). Tissue specimen evaluation did not
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show any signs of mucosal layer disruption or increased infiltration of inflammatory
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cells in any of the experimental groups.
184 3.2. Bacterial composition of the gut
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To determine possible changes in bacterial composition after low-dose of penicillin
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treatment, we detected gut taxonomic groups by 16S rRNA sequencing. We looked
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at the microbiota composition in the control and study group in gut tissue (distal
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ileum and ascending colon) and faecal samples (obtained directly form caecum
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lumen). Surprisingly, independently of treatment regimen (control or penicillin), we
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found substantial diversity of microbiota both at phyla and species level between
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littermates of C57BL6/J/cmdb male mice living in the same cage. Despite exactly the
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same environmental conditions (drinking water, chow diet, bedding, air ventilation
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system) microbiota of littermates at phyla level differed up to 40% for Firmicutes and
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Barcteroidetes (Figure 2A). This phenomenon was found both in control and study
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mice. Similar observations were made at other taxonomic levels, including species.
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(Figure 2B). Next, we evaluated the effect of low-dose of penicillin on the gut
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microbiota composition in comparison to control group without antibiotic. We did not
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find any significant changes in the presence of two major phyla groups – Firmicutes
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and Bacteroidetes (representing ~ 90% of gut microbiota) after penicillin treatment in
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small intestine, colon or fecal specimens (Figure 3A, B). Similarly, we did not find
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any significant alterations in other, less abundant, phyla of gut microbiota in
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evaluated specimens (data not shown). Interestingly, at the species taxonomic level,
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we found significant increase of bacteria Parabacteroides goldsteinii in stool and
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tissue samples from large intestine after penicillin treatment, in comparison to control
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group (9.5% vs. 4.9%, p=0.008 and 10.7% vs. 6.1%, p=0.008, respectively) (Figure
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3C). Similar increase of P. goldsteinii presence after antibiotic exposure was
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observed in small intestine tissue samples, however, in this case the results did not
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reach statistical significance (19.6% vs. 12.6%, p=0.098).
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3.3. Lymphocyte subsets in peripheral blood
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To determine whether penicillin affects the lymphocyte subpopulations, we
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performed flow cytometry on peripheral blood mononuclear cells (PBMC) from mice
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in both groups. Comparing to controls, mice treated with penicillin demonstrated
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significantly elevated percentage of B cells (CD19) (median 10.5% vs 8.0%, p=0.01)
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and decreased percentage of total CD4+ (median 75.4% vs 82.5%, p=0.0039),
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(Table 2). However among subsets of CD4+ lymphocytes, increased percentage of
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CD4+CD25+CD127low (Treg) cells (median 14.5% vs 8.1%, p=0.002) and CD4+
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cells producing IL-4 (Th2) (median 10.7% vs 7.8%, p=0.03) was detected in mice
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treated with penicillin. No significant differences in CD4+IL-17+ (TH17) cells (median
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4.9% vs 3.5%) or CD4+IFNγ+ (Th1) (median 7.9% vs 6.6%) were observed between
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groups.
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3.4. Lymphocyte subsets in gut tissue
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We also evaluated if changes in blood lymphocyte subpopulations after penicillin
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treatment were accompanied by any alterations in lymphocyte subsets infiltration in
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small or large intestine. Interestingly, immunohistochemical staining of small
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intestine and colon revealed no abnormalities or differences in lymphocyte subsets
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(CD3+, CD4+, CD8+, CD20+) between control and study group (Figure 4).
229 4. DISCUSSION
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Despite relatively stable composition of the gut microbiome in adult healthy
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individuals, there is an extremely high interpersonal diversity [4]. Data from Human
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Microbiome Project showed clear differences in Bacteroides/Firmicutes (two most
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abundant phyla in gut) ratio in healthy volunteers [13]. Similar observations were
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found at lower taxonomic units, like species [13, 14]. The cause and reason for this
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variability is currently unknown, however it may be driven by genetic factors,
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environmental factors (i.e. lifestyle and geographical differences between European,
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American and Asian population), diet or drugs (i.e. antibiotics) [4, 15]. The key
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question is, if the differences in the gut microbiome composition may predispose to
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the development of the diseases in selected persons. In our study, we have found
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significant differences in gut microbiome composition between mice. Surprisingly,
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this general phenomenon was independent of the treatment regimen. In our study,
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the animals were kept in specific pathogen-free conditions, with individually
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ventilated cages. They received the same autoclaved water and chow diet.
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Littermate mice were at the same age, freshly weaned from mother. Nevertheless,
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inter-animal differences in gut microbiome composition were still observed. This may
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suggest that genetic heterogeneity between mice is a driving force of microbiota
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composition in the case of identical housing (location, diet) and treatment (penicillin
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badge and dose) conditions. Of course, we cannot completely rule out that mouse
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husbandry condition influenced intestinal microbial composition, as it has been
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shown recently [16]. However, reported risk factors of inter-animals microbial
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differences and diversity, namely irradiated chow diet, open cages housing system,
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presence of other strains at the same room, or low restrictive access policy were not
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a case in our study. In our case, we compared microbiota of mice kept in the same
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animal facility, room and even a cage, so the effect of husbandry policy on microbial
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composition is highly unlikely.
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Another important finding of our study was significant increase of
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Parabacteroides goldsteinii in microbiota composition after penicillin exposure. P.
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goldsteinii is a Gram-negative, strictly anaerobic, rod-shaped bacteria described for
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the first time in 2005 [17]. Interestingly, P. goldsteinii can demonstrate resistance to
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many commonly used antibiotics like penicillin, clindamycin, erythromycin,
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piperacillin-tazobactam, meropenem, and may possess metallo-β-lactamase [18,
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19]. The bacteria is a part of human gut microbiota, but it was also isolated from
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abdominal abscesses and peritoneal fluid as a consequence of diverticulitis, caecal
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inflammation, and necrotic ileum or from appendix tissue in the case of acute
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appendicitis [17]. In our study, 4 weeks of mice exposure to low-doses of penicillin
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was sufficient to significantly increase population of P. goldsteinii in the colon and
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stool. Penicillin is a narrow spectrum antibiotic, more effective against Gram-positive
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bacteria, which may explain selective increase of specific Gram-negative pathogens.
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Elevated population of P. goldsteinii was accompanied by the increased percentage
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of neutrophils in peripheral blood of antibiotic treated mice, comparing to control.
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However, it is difficult to ascertain if this phenomenon was the direct effect of
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penicillin or pathogenic bacteria. The relationship between gut microbiome composition and function of
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immune system is already known, but it still remains under close evaluation [11, 20,
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21]. It has been recently shown that short-term treatment of mice with low-dose
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penicillin started from the birth, has altered metabolism and enhanced the risk of
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obesity [11]. This mechanism was, at least partially, mediated by changes in
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expression of ileal genes involved in immunity. Low-dose penicillin decreased
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expression of genes linked to antigen-presenting cells, T cells, B cells, and
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phagocytic cells. To investigate the immune cells shifts in response to an altered
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intestinal microbiota, we used murine model treated with low-dose of penicillin. Our
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data did not show any histological abnormalities in intestinal tissues or other organs
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of gastrointestinal tract like liver and pancreas (data not shown) in study group
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comparing to control. Additionally, we found no significant alterations among
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lymphocyte subsets in intestinal specimens after antibiotic exposure comparing to
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control. One of the possible explanation to these contradictory results is the use of
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different method for detection of immune cells in tissue in our study.
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It was previously reported, that influence of gut microbiota on the T cell subsets’
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balance extended beyond the intestinal lamina propria [22]. However, only limited
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studies were focused on the relationship between intestinal flora alteration and
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modification of peripheral blood immune cells. Banck et al. tested in vitro the effect
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of 20 different antibiotics on function of lymphocytes isolated from the peripheral
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blood, showing no influence of penicillin [20]. In our study, penicillin exposure
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resulted in the significant decrease of total CD4+ cells in blood. Similar pattern of
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lymphocyte counts, with decreased counts of CD4+ cells in lymphoid organs, but the
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same numbers of CD8+ and CD19+ as conventional colonized mice, was reported in
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germ-free mice [23]. However, in this study lymphocytes were isolated from the
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spleen, what has not been the case in our study. Low CD4+ count could be also the
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effect of dysbiosis caused by penicillin, since some bacteria, like Bacteroides fragilis,
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may affect the number of circulating CD4+ T cells [23]. Likewise, expansion of lamina
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propria and systemic Treg cells depends on intestinal microbiota, mainly on the
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presence of Clostridial strains [22]. In our study we were unable to identify the
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specific lymphocyte subpopulation responsible for the decrease of total CD4+
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lymphocytes. Unfortunately, selected CD4+ subpopulations representing only a small
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fraction of total CD4+ lymphocytes showed the opposite trend in the low-dose of
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penicillin group. Penicillin treated mice demonstrated increase of peripheral Treg
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(CD4+CD25+CD127low), as well as Th1 (IFNγ), Th2 (IL-4) cells, and B cells,
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although no functional studies were performed and we could not determine if this
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effect was caused by penicillin itself or indirectly by microbial imbalance,
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represented by increase of some specific bacterial strains, like Parabacteroides
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goldsteinii. Enhanced Th2 response in our study could be related to allergic reaction
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triggered by penicillin, since relationship between treatment with broad-spectrum
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antibiotics and exaggerated Th2 cell responses with increased circulating basophil
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populations, elevated IgE and allergic inflammation in mice was already reported
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[24]. On the other hand, penicillin has been also shown as an exacerbating factor of
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experimental autoimmune encephalomyelitis, the Th1 derived disease [25]. In this
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study, among evaluated beta-lactams, cefuroxime exposure resulted in down-
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regulation of genes expression associated with Th2 and Treg differentiation.
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Unfortunately, the effect of penicillin on these genes was not explored. However, it
321
was shown, that penicillin-modified albumin was able to enter T cells, and enhanced
322
their
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encephalomyelitis. [25]. In our study, penicillin exposure also resulted in significant
324
increase of Th1 (CD4+IFNγ+) lymphocytes as it was mentioned above. It is difficult to
325
speculate which subset of T cells dominated at the beginning of treatment and which
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of these cells would dominate after longer (i.e. several months) exposure to
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penicillin. Certainly, more research is needed to determine specific effects of
328
antibiotics on function of immune cells, both in peripheral blood and locally in
329
tissues. Our study has some limitations, including small sample size. However, our
330
data were consistent and low number of animals does not seem to affect the final
331
results. Moreover, in our experiment both study and control Group consisted
332
exclusively of male mice, so we cannot exclude that the results are gender specific.
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Cox et al. have already shown that temporary exposure to low-dose penicillin may
334
have a sex-specific long term effect (metabolic alterations, changes in ileal
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expression of genes involved in immunity) only during a specific time window –
336
before the birth of the pups and throughout the weaning process [11]. In this study,
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there was no difference in body composition of older male and female mice after
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antibiotic exposure. In our experiment, we used mice after the weaning period, so
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the probability that the results were affected by specific gender is much lower. In
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agriculture, administration of low doses of antibacterial agents promotes the growth
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the
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experimental
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of farm animals independently of sex. We also did not look at the expression of
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intestinal or colonic genes involved in immune reaction. Moreover, our observation
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period was limited to 4 weeks only. It would be interesting to prolong the experiment
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until the development of changes in mice body weight or autoimmune-related
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diseases.
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In summary, the present work documents unique and significant effect of
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penicillin on B and T cells in peripheral blood of young mice. This effect may be
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mediated through changes in gut microbiota represented by the expansion of
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Parabacteroides goldsteinii. Altering immune cells at a young age may have a
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tremendous effect on future development of autoimmune or allergic diseases.
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351 Funding
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This research received grant from Medical University of Bialystok, Poland
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(N/ST/ZB/16/001/1151)
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Competing interests
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The authors declare no competing financial interests.
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Ethical approval
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All in vivo experiments were performed according to EU Directive 2010/63/EU and
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approved by the Local Committee for Experiments with the Use of Laboratory
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Animals, Bialystok, Poland.
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group.
Penicillin administration
Histological changes
156.5 (150-159.3) NS 232.2 (183-240) NS 247 (191-267.4) NS
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141.3 (134.3-157) 222 (208-231.2) 251 (237-279.2)
2.56 (1.96-3.41) 95.5 (92-97) 4 (3-5) 776.5 (531-897) 9.1 (8.1-9.6) 13.8 (12.6-14.3) 44.4 (41.6-46.3) 48.5 (46.1-51.6)
2.58 (1.07-3.74) 92.5 (90-94) 6 (4-7) 773 (256-905) 9.2 (8.7-9.8) 13.8 (13.1-14.5) 44.6 (42-48.6) 48.4 (46-50.7)
no inflammation
no inflammation
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% Change of body weight (range) Day 7 Day 14 Day 28 CBC value median (min-max): WBC x 103/uL Lymphocytes (% of WBC) Neutrophils (% of WBC) Platelets x 103/uL RBC x 106/uL Hb (g/dl) Ht (%) MCV (fl)
p
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Control
NS 0.0002 0.0002 NS NS NS NS NS NS
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CBC – complete blood count, WBC – white blood count, Hb – haemoglobin, Ht – haematocrit, MCV – mean corpuscular volume
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88.1 (74.2-93.2) 82.5 (77.6-92.7) 24.7 (13.2-26.9) 8.1 (3.8-12.9) 21.7 (17.8-31.4) 7.8 (7.4-9.2) 6.6 (5.5-16.3) 3.5 (2.4-4.7) 8.0 (4.9-11.2) 60.7 (56.8-61.4) 38.3 (35.5-40.4)
81.7(65.2-89.6) 75.4 (62.1-80.9) 28.7 (21-39.3) 14.5 (6.5-17.8) 28.2 (25.2-31.3) 10.7 (7.8-12.3) 7.9 (5.8-11) 4.9 (4.7-5.2) 10.5 (8.1-13.2) 55.2 (53.8-57.4) 42.1 (37.6 – 43.8)
0.029 0.0039 NS 0.002 0.007 0.03 NS 0.008 0.01 0.02 NS
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Penicillin administration median (min-max)
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CD3+ (%) CD4+ (%) CD8+ (%) CD4+CD25+CD127low (%) CD4+CD25+CD127high (%) CD4+ IL-4 (%) CD4+IL-17 (%) CD4+IFNγ (%) CD19+ (%) sIgM+CD45R+CD23- (%) sIgM+CD45R+CD23+ (%)
Control median (min-max)
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ig. 1. Representative H&E staining of small intestine (A) and colon (B) sections of mice after 28 days o
eatment with water vs. water plus penicillin. Histology evaluation revealed no abnormalities in mucosa of termina
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art of ileum (A) and ascending colon (B) in control and penicillin treated mice (control = 10, penicillin = 10).
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Fig. 2. Differences in gut microbiota between C57BL6/J/cmdb male mice kept in the same conditions. Essent
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variations in small intestine, colon and faecal microbiota composition at phyla (A) or species (B) taxonomic level in mic
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ittermates were observed. Five circles in each square represent five separate mice housed in the same cage. Difference
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n microbiota between littermates were observed both in control and penicillin treated mice.
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Fig. 3. Changes in small intestine, colon and faecal microbiota composition after 28 days of penicillin
treatment. At the phyla taxonomic level there was no significant difference in Firmicutes (A) and Bacteroidetes (B)
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presence between control and penicillin treated mice. At the species taxonomic level significant increase of
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Parabacteroides goldsteinii (C) after penicillin treatment was observed. Each bar represents average from five
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Fig. 4. Representative immunohistochemistry staining of small intestine sections of mice after 28 days of penicillin treatment. Histology evaluation revealed no differences in intestinal infiltration of lymphocytes T CD3+ (A),
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CD4+ (B) and CD8+ (C) and lymphocytes B CD20+ (D) (control = 10, penicillin = 10).
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Murine gut microbiota is highly variable between littermate mice Penicillin treatment increases abundance of pathogenic P. goldsteinii bacteria Antibiotic treatment has a profound effect on the immune system
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• • •
S1075-9964(17)30058-6
DOI:
10.1016/j.anaerobe.2017.03.015
Reference:
YANAE 1711
To appear in:
Anaerobe
Received Date: 24 December 2016 Revised Date:
4 March 2017
Accepted Date: 15 March 2017
Please cite this article as: Daniluk J, Daniluk U, Rusak M, Dabrowska M, Reszec J, Garbowicz M, Humińska K, Dabrowski A, The effect of penicillin administration in early life on murine gut microbiota and blood lymphocyte subsets, Anaerobe (2017), doi: 10.1016/j.anaerobe.2017.03.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The effect of penicillin administration in early life on murine gut microbiota
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and blood lymphocyte subsets.
3 Jaroslaw Daniluk1, Urszula Daniluk2, Malgorzata Rusak3, Milena Dabrowska3,
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Joanna Reszec4, Magdalena Garbowicz5, Kinga Humińska5, Andrzej Dabrowski1
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Bialystok ul. M. Sklodowskiej-Curie 24a, 15-276 Bialystok, Poland
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Department of Gastroenterology and Internal Medicine, Medical University of
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Department of Pediatrics, Gastroenterology and Allergology, Medical University of
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Bialystok ul. J. Waszyngtona 17, 15-274 Bialystok, Poland
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3
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Waszyngtona 15A, 15-269 Bialystok, Poland
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Waszyngtona 13, 15-269 Bialystok, Poland
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Poland
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Department of Medical Pathomorphology, Medical University of Bialystok ul. J.
Genomic Laboratory, DNA Research Center ul. Mickiewicza 31, 60-385 Poznan,
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Department of Haematological Diagnostics, Medical University of Bialystok ul. J.
Corresponding author: Jaroslaw Daniluk, Department of Gastroenterology and
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Internal Medicine, Medical University of Bialystok ul. M. Sklodowskiej-Curie 24a, 15-
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276 Bialystok, Poland
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Phone: 48 85 746 82 34
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fax: 48 85 746 85 06
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Email: [email protected], [email protected]
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Abstract Background and aim: Antibiotics have many beneficial effects but their
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uncontrolled use may lead to increased risk of serious diseases in the future. Our
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hypothesis is that an early antibiotic exposition may affect immune system by
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altering gut microbiota. Therefore, the aim of the study was to determine the effect of
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penicillin treatment on gut microorganisms and immune system of mice. Methods:
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21-days old C57BL6/J/cmdb male mice were treated with low-dose of penicillin
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(study group) or water only (control group) for 4 weeks. Tissue and stool samples for
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histology or microbiome assessment and peripheral blood for CBC and flow
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cytometry evaluation were collected. Results: We found high variability in microbiota
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composition at different taxonomic levels between littermate mice kept in the same
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conditions, independently of treatment regimen. Interestingly, low-dose of penicillin
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caused significant increase of Parabacteroides goldsteinii in stool and in colon tissue
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in comparison to control group (9.5% vs. 4.9%, p=0.008 and 10.7% vs. 6.1%,
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p=0.008, respectively). Moreover, mice treated with penicillin demonstrated
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significantly elevated percentage of B cells (median 10.5% vs 8.0%, p=0.01) and
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decrease in the percentage of total CD4+ cell (median 75.4% vs 82.5%, p=0.0039)
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with subsequent changes among subsets - increased percentage of regulatory T
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cells (Treg), T helper 1 (Th1) and T helper 2 (Th2) cells. Conclusion: Our study
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showed significant effect of penicillin on B and T cells in peripheral blood of young
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mice. This effect may be mediated through changes in gut microbiota represented
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by the expansion of Parabacteroides goldsteinii.
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Key words: lymphocytes, microbiota, Parabacteroides goldsteinii, penicillin
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1. Introduction Discovery of penicillin in 1928 by Sir Alexander Fleming, saved many lives
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and revolutionized treatment of bacterial infections. Antibiotics have also found
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application in agriculture, due to their effect on weight gain (up to 15%) in farm
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animals [1]. Nowadays easy access to the antibiotics led to their overuse in humans
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and caused development of bacterial resistance to the treatment.
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Despite of their benefit in infection treatment, antibiotics have profound
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influence on the structure and function of gut microbes. Our knowledge about human
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microbiome has dramatically increased over the last decade due to development of
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novel sequence-based molecular tests, like 16s rRNA-based sequencing, which
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became the gold standard technique in microbiome identification [2]. Commensal
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microorganisms prevent colonization of the gut by pathogens. They are involved in
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the process of appropriate development and maturation of innate and acquired
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immune system during the childhood, and maintaining homeostasis in the adults [3,
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4]. The fetal gastrointestinal tract is sterile but immediately after the birth it is
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colonized by many different microorganisms [5]. Recently, it has been reported that
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any perturbations in microorganisms composition caused by antibiotics or changes
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in the diet habits during the first two years of live, may predispose to the
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development of chronic diseases like obesity, type 1 diabetes, rheumatoid arthritis,
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coeliac disease, liver diseases and inflammatory bowel disease (IBD) [6-8]. It has
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been also suggested that antibiotic exposure during the first year of life increases
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the risk of asthma development [9]. Jernberg et al reported that even a short, 1-week
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long course of antibiotic regimen causes significant and prolonged (up to 2 years)
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changes in microbiota composition [10]. Therefore, the aim of our study was to
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determine the effect of early antibiotic (penicillin) exposition on gut microorganisms
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and immune system of mice.
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74 2. Material and methods 2.1. Animals
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C57BL6/J/cmdb male mice (Medical University of Bialystok; Bialystok, Poland) were
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maintained in specific pathogen-free (SPF) conditions. All in vivo experiments were
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performed according to EU Directive 2010/63/EU and approved by the Local
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Committee for Experiments with the Use of Laboratory Animals, Bialystok, Poland.
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21-days old mice, just after the weaning, were randomly divided into two groups:
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study group – animals receiving penicillin dissolved in drinking water (n=10) and
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control group – mice receiving drinking water only (n=10), ad libitum for 28 days.
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Dose of the antibiotic, 1 µg of penicillin / 1 gram of body weight, was determined on
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the basis of previous literature data and assumption that daily consumption of water
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is 15 ml per 100 g of mice [11, 12]. Fresh containers with water and water with
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antibiotic were changed three times a week. Mice were housed 5 per cage and they
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were allowed to eat standard chow diet ad libitum. Consumption of water or water
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with penicillin was determined on daily basis for each cage. Body weight of each
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mouse was measured every fourth day.
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2.2. Sample Collection
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After 28 days, mice were anesthetized and sacrificed. Blood samples were collected
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by cardiac puncture and divided into two parts - complete blood count (CBC) and
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flow cytometry analysis. The small and large intestine, pancreas, and liver were
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harvested aseptically and stored for further histological evaluation. For microbiota
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assessment samples were collected directly from tissue (distal part of small intestine
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and ascending colon), and from the stool (lumen of the caecum), and immediately
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snap-frozen and stored (-80°C).
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2.3. CBC and flow cytometry analysis of isolated cells
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To determine CBC, 200 µl of peripheral blood was used and analyzed in the
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Department of Haematological Diagnostics, Medical University of Bialystok.
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Peripheral subpopulations of lymphocytes were assessed using flow cytometry. To
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prepare samples, approximately 200 µl of peripheral blood was treated with RBC
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lysis buffer (Sigma-Aldrich) for 10 min at room temperature, and the remaining cells
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were washed twice with cold PBS and centrifuged at 1,200 rpm for 10 min. Cells
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were stained with the appropriate combinations of the following antibodies: FITC–
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anti-CD3e (145-2C11; BD Pharmigen), APC-anti-CD4 (MR4-5; BD Pharmigen), PE-
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anti-CD25 (3C7; BD Pharmigen), PE-Cy7-anti-CD127 (SB/199; BD Pharmigen), and
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Mouse T Lymphocyte Subset Antibody Cocktail with Isotype Control (BD Pharmigen)
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containing PE-Cy 7- anti-CD3e (145-2C11), PE-anti-CD4 (RM4-5), APC-anti- CD8a
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(53-6.7), Mouse B Lymphocyte Subset Antibody Cocktail with Isotype Control (BD
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Pharmigen) with PE-Cy 7- anti-CD45R/B220 (145-2C11); PE-anti-CD23 (RM4-5);
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APC-anti- sIgM (53-6.7). The Mouse Th1/Th2/Th17 Phenotyping Kit (PerCP-Cy5.5-
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anti-CD4 (RM4-5); PE-anti-IL-17A (TC11-18H10.1); FITC-anti-INF-GMA (XMG1.2);
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APC-anti-CD4 (11B11) BD Pharmigen) were used according to the manufacturer’s
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instruction for the detection of CD4+IL-17+, CD4+IFNγ+, CD4+IL-4+ expression.
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Flow cytometric data were acquired using a FACS Canto II cytometer with BD
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FACSDiva Software v6.1.3 (BD Biosciences) and analyzed with Worksheet software
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(BD).
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2.4. Histology and Immunohistochemistry
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The organs were fixed with 10 % PBS-buffered formalin for 24h, embedded in
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paraffin, cut sagittally into 5-µm sections, stained with hematoxylin and eosin (H&E),
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and examined by light microscopy (Olympus BX45) for histological analysis. For
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each animal, ten fields at a magnification of ×100 were captured randomly from the
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four different parts of the intestine.
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To determinate the lymphocytic infiltration we used antibodies against T cytotoxic
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cells, T helpers and against lymphocytes B. Following the deparaffinization and
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rehydration, epitope retrieval was carried out in the EnVision Flex Target Retrieval
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Solution (DAKO) at low pH. Endogenous peroxidases were blocked by incubating
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the sections in methanol and 3% hydrogen peroxidase for 40 minutes. Next, slides
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were incubated with proper anti-mouse antibodies against CD3, CD4, CD8 and
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CD20 in 1:100 dilutions for 1 hour at room temperature. Visualization reagent
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EnVision (DAKO) was applied for 30 minutes and followed by DAB solution for 10
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minutes. The slides were than counterstained with H&E and examined under the
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light microscope. The intensity of immunostaining was evaluated in random 10 fields
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under ×20 magnification. Appropriate positive and negative controls were performed.
140 2.5. Faecal microbiota analysis
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The bacterial composition of small and large intestine, and stool samples of each
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group was carried out using 16S ribosomal RNA (rRNA) gene sequencing on the
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MiSeq apparatus (Illumina) via next-generation sequencing (NGS) technology. In
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order to analyze bacterial components, the V3-V4 hypervariable region of the 16S
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rRNA gene was firstly amplified from the genomic DNA extracted from fecal samples
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by
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GACTACHVGGGTATCTAATCC-3’)
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sequences, compatible with Illumina’s indices. Incorporation of primers with indexes
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and adapters was followed by second PCR reactions. After each amplification step,
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the PCR products were purified by using AMPure XP beads (Beckman Coulter
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Genomic, CA, USA). DNA amplified fragments were normalized to 2nM by Qubit®
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dsDNA HS kit (high
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sequencing. 8pM libraries were loaded on the MiSeq plarform (Illumina Inc., San
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Diego, CA, USA). Sequencing was performed with 300-nucleotide-long paired-end
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reads on the Illumina sequencer (MiSeq) according to the instructions of the
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manufacturer. The analysis output file generated for the 16S metagenomics
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workflow provided classification of reads for each sample. Obtained data were
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analyzed and interpreted using specialized software packages: Quantitative Insights
primers:
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(5’-CCTACGGGNGGCWGCAG-3’) containing
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into Microbial Ecology (QIIME), Greengenes, Metagenome Analyzer (MEGAN) I
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Ribosomal Database Project (RDP).
162 2.6. Data analysis
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Data were analyzed using Statistica 10 software. Statistical significance was
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determined by U Mann-Whitney test; p<0.05 was considered statistically significant.
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166 3. Results
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3.1. Body weight, CBC and histological evaluation
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During the whole study period, body weights of animals were within the range of
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growth for C57BL6/J/cmdb male mice. There was no statistically significant
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difference in body weight change between control and study groups (Table 1). There
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was no difference in consumption of water in control (mean 21.8 ml/cage/day) or
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water with penicillin in study group (mean 23.0 ml/cage/day). After 28 days of
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penicillin administration no differences in white blood count, red blood count or
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platelets count were observed between the groups. However, 4 weeks of antibiotic
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exposure significantly increased percentage of blood neutrophils in comparison to
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control group (6.0% vs. 4.0%, p=0.0002). This was accompanied by significant
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decrease of lymphocytes percentage in the treatment group in comparison to control
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(92.5% vs. 95.0% respectively, p=0.0002).
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Histological examination of the small and large intestinal biopsies revealed no
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abnormalities between study groups (Fig. 1). Tissue specimen evaluation did not
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show any signs of mucosal layer disruption or increased infiltration of inflammatory
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cells in any of the experimental groups.
184 3.2. Bacterial composition of the gut
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To determine possible changes in bacterial composition after low-dose of penicillin
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treatment, we detected gut taxonomic groups by 16S rRNA sequencing. We looked
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at the microbiota composition in the control and study group in gut tissue (distal
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ileum and ascending colon) and faecal samples (obtained directly form caecum
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lumen). Surprisingly, independently of treatment regimen (control or penicillin), we
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found substantial diversity of microbiota both at phyla and species level between
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littermates of C57BL6/J/cmdb male mice living in the same cage. Despite exactly the
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same environmental conditions (drinking water, chow diet, bedding, air ventilation
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system) microbiota of littermates at phyla level differed up to 40% for Firmicutes and
195
Barcteroidetes (Figure 2A). This phenomenon was found both in control and study
196
mice. Similar observations were made at other taxonomic levels, including species.
197
(Figure 2B). Next, we evaluated the effect of low-dose of penicillin on the gut
198
microbiota composition in comparison to control group without antibiotic. We did not
199
find any significant changes in the presence of two major phyla groups – Firmicutes
200
and Bacteroidetes (representing ~ 90% of gut microbiota) after penicillin treatment in
201
small intestine, colon or fecal specimens (Figure 3A, B). Similarly, we did not find
202
any significant alterations in other, less abundant, phyla of gut microbiota in
203
evaluated specimens (data not shown). Interestingly, at the species taxonomic level,
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we found significant increase of bacteria Parabacteroides goldsteinii in stool and
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tissue samples from large intestine after penicillin treatment, in comparison to control
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group (9.5% vs. 4.9%, p=0.008 and 10.7% vs. 6.1%, p=0.008, respectively) (Figure
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3C). Similar increase of P. goldsteinii presence after antibiotic exposure was
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observed in small intestine tissue samples, however, in this case the results did not
209
reach statistical significance (19.6% vs. 12.6%, p=0.098).
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3.3. Lymphocyte subsets in peripheral blood
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To determine whether penicillin affects the lymphocyte subpopulations, we
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performed flow cytometry on peripheral blood mononuclear cells (PBMC) from mice
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in both groups. Comparing to controls, mice treated with penicillin demonstrated
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significantly elevated percentage of B cells (CD19) (median 10.5% vs 8.0%, p=0.01)
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and decreased percentage of total CD4+ (median 75.4% vs 82.5%, p=0.0039),
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(Table 2). However among subsets of CD4+ lymphocytes, increased percentage of
218
CD4+CD25+CD127low (Treg) cells (median 14.5% vs 8.1%, p=0.002) and CD4+
219
cells producing IL-4 (Th2) (median 10.7% vs 7.8%, p=0.03) was detected in mice
220
treated with penicillin. No significant differences in CD4+IL-17+ (TH17) cells (median
221
4.9% vs 3.5%) or CD4+IFNγ+ (Th1) (median 7.9% vs 6.6%) were observed between
222
groups.
223
3.4. Lymphocyte subsets in gut tissue
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We also evaluated if changes in blood lymphocyte subpopulations after penicillin
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treatment were accompanied by any alterations in lymphocyte subsets infiltration in
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small or large intestine. Interestingly, immunohistochemical staining of small
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intestine and colon revealed no abnormalities or differences in lymphocyte subsets
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(CD3+, CD4+, CD8+, CD20+) between control and study group (Figure 4).
229 4. DISCUSSION
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Despite relatively stable composition of the gut microbiome in adult healthy
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individuals, there is an extremely high interpersonal diversity [4]. Data from Human
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Microbiome Project showed clear differences in Bacteroides/Firmicutes (two most
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abundant phyla in gut) ratio in healthy volunteers [13]. Similar observations were
235
found at lower taxonomic units, like species [13, 14]. The cause and reason for this
236
variability is currently unknown, however it may be driven by genetic factors,
237
environmental factors (i.e. lifestyle and geographical differences between European,
238
American and Asian population), diet or drugs (i.e. antibiotics) [4, 15]. The key
239
question is, if the differences in the gut microbiome composition may predispose to
240
the development of the diseases in selected persons. In our study, we have found
241
significant differences in gut microbiome composition between mice. Surprisingly,
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this general phenomenon was independent of the treatment regimen. In our study,
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the animals were kept in specific pathogen-free conditions, with individually
244
ventilated cages. They received the same autoclaved water and chow diet.
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Littermate mice were at the same age, freshly weaned from mother. Nevertheless,
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inter-animal differences in gut microbiome composition were still observed. This may
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suggest that genetic heterogeneity between mice is a driving force of microbiota
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composition in the case of identical housing (location, diet) and treatment (penicillin
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badge and dose) conditions. Of course, we cannot completely rule out that mouse
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husbandry condition influenced intestinal microbial composition, as it has been
251
shown recently [16]. However, reported risk factors of inter-animals microbial
252
differences and diversity, namely irradiated chow diet, open cages housing system,
253
presence of other strains at the same room, or low restrictive access policy were not
254
a case in our study. In our case, we compared microbiota of mice kept in the same
255
animal facility, room and even a cage, so the effect of husbandry policy on microbial
256
composition is highly unlikely.
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Another important finding of our study was significant increase of
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Parabacteroides goldsteinii in microbiota composition after penicillin exposure. P.
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goldsteinii is a Gram-negative, strictly anaerobic, rod-shaped bacteria described for
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the first time in 2005 [17]. Interestingly, P. goldsteinii can demonstrate resistance to
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many commonly used antibiotics like penicillin, clindamycin, erythromycin,
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piperacillin-tazobactam, meropenem, and may possess metallo-β-lactamase [18,
263
19]. The bacteria is a part of human gut microbiota, but it was also isolated from
264
abdominal abscesses and peritoneal fluid as a consequence of diverticulitis, caecal
265
inflammation, and necrotic ileum or from appendix tissue in the case of acute
266
appendicitis [17]. In our study, 4 weeks of mice exposure to low-doses of penicillin
267
was sufficient to significantly increase population of P. goldsteinii in the colon and
268
stool. Penicillin is a narrow spectrum antibiotic, more effective against Gram-positive
269
bacteria, which may explain selective increase of specific Gram-negative pathogens.
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Elevated population of P. goldsteinii was accompanied by the increased percentage
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of neutrophils in peripheral blood of antibiotic treated mice, comparing to control.
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However, it is difficult to ascertain if this phenomenon was the direct effect of
273
penicillin or pathogenic bacteria. The relationship between gut microbiome composition and function of
275
immune system is already known, but it still remains under close evaluation [11, 20,
276
21]. It has been recently shown that short-term treatment of mice with low-dose
277
penicillin started from the birth, has altered metabolism and enhanced the risk of
278
obesity [11]. This mechanism was, at least partially, mediated by changes in
279
expression of ileal genes involved in immunity. Low-dose penicillin decreased
280
expression of genes linked to antigen-presenting cells, T cells, B cells, and
281
phagocytic cells. To investigate the immune cells shifts in response to an altered
282
intestinal microbiota, we used murine model treated with low-dose of penicillin. Our
283
data did not show any histological abnormalities in intestinal tissues or other organs
284
of gastrointestinal tract like liver and pancreas (data not shown) in study group
285
comparing to control. Additionally, we found no significant alterations among
286
lymphocyte subsets in intestinal specimens after antibiotic exposure comparing to
287
control. One of the possible explanation to these contradictory results is the use of
288
different method for detection of immune cells in tissue in our study.
289
It was previously reported, that influence of gut microbiota on the T cell subsets’
290
balance extended beyond the intestinal lamina propria [22]. However, only limited
291
studies were focused on the relationship between intestinal flora alteration and
292
modification of peripheral blood immune cells. Banck et al. tested in vitro the effect
293
of 20 different antibiotics on function of lymphocytes isolated from the peripheral
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blood, showing no influence of penicillin [20]. In our study, penicillin exposure
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resulted in the significant decrease of total CD4+ cells in blood. Similar pattern of
296
lymphocyte counts, with decreased counts of CD4+ cells in lymphoid organs, but the
297
same numbers of CD8+ and CD19+ as conventional colonized mice, was reported in
298
germ-free mice [23]. However, in this study lymphocytes were isolated from the
299
spleen, what has not been the case in our study. Low CD4+ count could be also the
300
effect of dysbiosis caused by penicillin, since some bacteria, like Bacteroides fragilis,
301
may affect the number of circulating CD4+ T cells [23]. Likewise, expansion of lamina
302
propria and systemic Treg cells depends on intestinal microbiota, mainly on the
303
presence of Clostridial strains [22]. In our study we were unable to identify the
304
specific lymphocyte subpopulation responsible for the decrease of total CD4+
305
lymphocytes. Unfortunately, selected CD4+ subpopulations representing only a small
306
fraction of total CD4+ lymphocytes showed the opposite trend in the low-dose of
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penicillin group. Penicillin treated mice demonstrated increase of peripheral Treg
308
(CD4+CD25+CD127low), as well as Th1 (IFNγ), Th2 (IL-4) cells, and B cells,
309
although no functional studies were performed and we could not determine if this
310
effect was caused by penicillin itself or indirectly by microbial imbalance,
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represented by increase of some specific bacterial strains, like Parabacteroides
312
goldsteinii. Enhanced Th2 response in our study could be related to allergic reaction
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triggered by penicillin, since relationship between treatment with broad-spectrum
314
antibiotics and exaggerated Th2 cell responses with increased circulating basophil
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populations, elevated IgE and allergic inflammation in mice was already reported
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[24]. On the other hand, penicillin has been also shown as an exacerbating factor of
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experimental autoimmune encephalomyelitis, the Th1 derived disease [25]. In this
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study, among evaluated beta-lactams, cefuroxime exposure resulted in down-
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regulation of genes expression associated with Th2 and Treg differentiation.
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Unfortunately, the effect of penicillin on these genes was not explored. However, it
321
was shown, that penicillin-modified albumin was able to enter T cells, and enhanced
322
their
323
encephalomyelitis. [25]. In our study, penicillin exposure also resulted in significant
324
increase of Th1 (CD4+IFNγ+) lymphocytes as it was mentioned above. It is difficult to
325
speculate which subset of T cells dominated at the beginning of treatment and which
326
of these cells would dominate after longer (i.e. several months) exposure to
327
penicillin. Certainly, more research is needed to determine specific effects of
328
antibiotics on function of immune cells, both in peripheral blood and locally in
329
tissues. Our study has some limitations, including small sample size. However, our
330
data were consistent and low number of animals does not seem to affect the final
331
results. Moreover, in our experiment both study and control Group consisted
332
exclusively of male mice, so we cannot exclude that the results are gender specific.
333
Cox et al. have already shown that temporary exposure to low-dose penicillin may
334
have a sex-specific long term effect (metabolic alterations, changes in ileal
335
expression of genes involved in immunity) only during a specific time window –
336
before the birth of the pups and throughout the weaning process [11]. In this study,
337
there was no difference in body composition of older male and female mice after
338
antibiotic exposure. In our experiment, we used mice after the weaning period, so
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the probability that the results were affected by specific gender is much lower. In
340
agriculture, administration of low doses of antibacterial agents promotes the growth
in
the
development
of
experimental
autoimmune
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of farm animals independently of sex. We also did not look at the expression of
342
intestinal or colonic genes involved in immune reaction. Moreover, our observation
343
period was limited to 4 weeks only. It would be interesting to prolong the experiment
344
until the development of changes in mice body weight or autoimmune-related
345
diseases.
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In summary, the present work documents unique and significant effect of
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penicillin on B and T cells in peripheral blood of young mice. This effect may be
348
mediated through changes in gut microbiota represented by the expansion of
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Parabacteroides goldsteinii. Altering immune cells at a young age may have a
350
tremendous effect on future development of autoimmune or allergic diseases.
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351 Funding
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This research received grant from Medical University of Bialystok, Poland
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(N/ST/ZB/16/001/1151)
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Competing interests
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The authors declare no competing financial interests.
357
Ethical approval
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All in vivo experiments were performed according to EU Directive 2010/63/EU and
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approved by the Local Committee for Experiments with the Use of Laboratory
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Animals, Bialystok, Poland.
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Penicillin administration
Histological changes
156.5 (150-159.3) NS 232.2 (183-240) NS 247 (191-267.4) NS
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141.3 (134.3-157) 222 (208-231.2) 251 (237-279.2)
2.56 (1.96-3.41) 95.5 (92-97) 4 (3-5) 776.5 (531-897) 9.1 (8.1-9.6) 13.8 (12.6-14.3) 44.4 (41.6-46.3) 48.5 (46.1-51.6)
2.58 (1.07-3.74) 92.5 (90-94) 6 (4-7) 773 (256-905) 9.2 (8.7-9.8) 13.8 (13.1-14.5) 44.6 (42-48.6) 48.4 (46-50.7)
no inflammation
no inflammation
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% Change of body weight (range) Day 7 Day 14 Day 28 CBC value median (min-max): WBC x 103/uL Lymphocytes (% of WBC) Neutrophils (% of WBC) Platelets x 103/uL RBC x 106/uL Hb (g/dl) Ht (%) MCV (fl)
p
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Control
NS 0.0002 0.0002 NS NS NS NS NS NS
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CBC – complete blood count, WBC – white blood count, Hb – haemoglobin, Ht – haematocrit, MCV – mean corpuscular volume
ACCEPTED MANUSCRIPT Table 2 Changes in the percentage of lymphocyte subsets in peripheral blood between study groups. Sample size was 10 mice per group.
p
88.1 (74.2-93.2) 82.5 (77.6-92.7) 24.7 (13.2-26.9) 8.1 (3.8-12.9) 21.7 (17.8-31.4) 7.8 (7.4-9.2) 6.6 (5.5-16.3) 3.5 (2.4-4.7) 8.0 (4.9-11.2) 60.7 (56.8-61.4) 38.3 (35.5-40.4)
81.7(65.2-89.6) 75.4 (62.1-80.9) 28.7 (21-39.3) 14.5 (6.5-17.8) 28.2 (25.2-31.3) 10.7 (7.8-12.3) 7.9 (5.8-11) 4.9 (4.7-5.2) 10.5 (8.1-13.2) 55.2 (53.8-57.4) 42.1 (37.6 – 43.8)
0.029 0.0039 NS 0.002 0.007 0.03 NS 0.008 0.01 0.02 NS
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Penicillin administration median (min-max)
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CD3+ (%) CD4+ (%) CD8+ (%) CD4+CD25+CD127low (%) CD4+CD25+CD127high (%) CD4+ IL-4 (%) CD4+IL-17 (%) CD4+IFNγ (%) CD19+ (%) sIgM+CD45R+CD23- (%) sIgM+CD45R+CD23+ (%)
Control median (min-max)
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ig. 1. Representative H&E staining of small intestine (A) and colon (B) sections of mice after 28 days o
eatment with water vs. water plus penicillin. Histology evaluation revealed no abnormalities in mucosa of termina
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art of ileum (A) and ascending colon (B) in control and penicillin treated mice (control = 10, penicillin = 10).
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Fig. 2. Differences in gut microbiota between C57BL6/J/cmdb male mice kept in the same conditions. Essent
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variations in small intestine, colon and faecal microbiota composition at phyla (A) or species (B) taxonomic level in mic
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ittermates were observed. Five circles in each square represent five separate mice housed in the same cage. Difference
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n microbiota between littermates were observed both in control and penicillin treated mice.
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Fig. 3. Changes in small intestine, colon and faecal microbiota composition after 28 days of penicillin
treatment. At the phyla taxonomic level there was no significant difference in Firmicutes (A) and Bacteroidetes (B)
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presence between control and penicillin treated mice. At the species taxonomic level significant increase of
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Parabacteroides goldsteinii (C) after penicillin treatment was observed. Each bar represents average from five
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Fig. 4. Representative immunohistochemistry staining of small intestine sections of mice after 28 days of penicillin treatment. Histology evaluation revealed no differences in intestinal infiltration of lymphocytes T CD3+ (A),
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CD4+ (B) and CD8+ (C) and lymphocytes B CD20+ (D) (control = 10, penicillin = 10).
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Murine gut microbiota is highly variable between littermate mice Penicillin treatment increases abundance of pathogenic P. goldsteinii bacteria Antibiotic treatment has a profound effect on the immune system
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