Gut microbiota recovery and immune response in ampicillin-treated mice

Accepted Manuscript Gut microbiota recovery and immune response in ampicillintreated mice

Josué L. Castro-Mejía, Maja Jakesevic, Niels F. Fabricius, Łukasz Krych, Dennis S. Nielsen, Witold Kot, Katja M. Bendtsen, Finn K. Vogensen, Camilla H.F. Hansen, Axel K. Hansen PII: DOI: Reference:

S0034-5288(18)30010-9 doi:10.1016/j.rvsc.2018.03.013 YRVSC 3547

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

5 January 2018 22 March 2018 24 March 2018

Please cite this article as: Josué L. Castro-Mejía, Maja Jakesevic, Niels F. Fabricius, Łukasz Krych, Dennis S. Nielsen, Witold Kot, Katja M. Bendtsen, Finn K. Vogensen, Camilla H.F. Hansen, Axel K. Hansen , Gut microbiota recovery and immune response in ampicillin-treated mice. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yrvsc(2018), doi:10.1016/ j.rvsc.2018.03.013

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Gut microbiota recovery and immune response in ampicillin-treated mice Josué L Castro-Mejíaa* , Maja Jakesevic b* , Niels F Fabriciusb , Łukasz Krycha, Dennis S Nielsena,

a

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Witold Kotc, Katja M Bendtsenb , Finn K Vogensena, Camilla H F Hansenb , Axel K Hansenb# Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26,

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1958 Frederiksberg, Denmark, [email protected], [email protected], [email protected],

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences,

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b

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[email protected]

University of Copenhagen, Thorvaldsensvej 57, 1870 Frederiksberg, Denmark,

[email protected], [email protected]

Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000

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[email protected], [email protected], [email protected],

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Roskilde, Denmark, [email protected]

These authors contributed equally to this work

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Corresponding author: Axel K Hansen, e-mail: [email protected]

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*

'Declarations of interest: ‘none'.

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Abstract Ampicillin is applied in rodents to induce a temporarily depleted microbiota. To elucidate whether bacteria are just temporarily suppressed or fully eliminated, and how this affects the recolonisation process, we compared the microbiota and immune system in conventionally housed untreated mice with newly weaned ampicillin treated mice subsequently housed in either a

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microbe containing environment or in an isolator with only host associated suppressed bacteria

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to recolonize the gut. Two weeks ampicillin treatment induced a seemingly germ-free state with no bacterial DNA to reveal. Four weeks after treatment caeca were still significantly enlarged in

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both treated groups, but bacteria re-appeared even in isolator housed mice. While some

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suppressed bacteria were able to recover and even dominate the community, the abundances and composition were far from the untreated mice and differed between isolator and conventional

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housing. The treatment reduced the innate cytokine expressions at least for three weeks after treatment, and had a non-lasting reducing impact on the regulatory T cells, and a more lasting impact on the natural killer T cells. We conclude that temporary ampicillin treatment suppresses

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the majority but does not eliminate all the gut microbiota members. The re-colonisation process

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is as such influenced by both suppressed host associated bacteria and by environmental bacteria. Treated mice do not re-obtain a complex gut microbiota comparable to untreated mice, and the immune response and gut morphology reflect this. This is a concern when comparing host

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parameters sensitive to microbial regulation after an antibiotic-induced temporarily ‘germ-free’

Keywords: Microbiota, mice, ampicillin, environment, immune system

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Introduction Over the last decade increasing numbers of studies have focused on the gut microbiota (GM) impact on disease development. Germ-free animals have been used extensively, and have shown that GM is essential for the growth, development, and function of the gastrointestinal tract (Berg, 1996; Hansen et al., 2012) . Microbial imbalance has in both humans and animal models

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been associated with an enhanced risk of different diseases, such as inflammatory bowel

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diseases (Holgersen et al., 2014; Marteau et al., 2004), allergy (Lundberg et al., 2012; Wang et al., 2008), obesity (Ley, 2010; Turnbaugh et al., 2006), and type 2 diabetes (Larsen et al., 2010;

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Rune et al., 2013). Germ-free animals are important tools for studying the impact of early life

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microbial colonisation on later life reactions of the immune system (Cebra, 1999; Hansen et al., 2012; Kelly et al., 2007). In germ-free mice, an early period of life without microbes induce

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long lasting effects on regulatory T cells, NKT cells and various cytokines characteristic of the Th2 dominated phenotype observed in germ-free mice (Hansen et al., 2012; Mazmanian and Kasper, 2006). However, the main disadvantage of germ-free animals is the inability to

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introduce a temporary germ-free status in mice harbouring a complex microbiota, which instead

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has been achieved by application of antibiotics. For example, ampicillin treatment limited to the early life of mice improves glucose tolerance during treatment (Bech-Nielsen et al., 2012; Membrez et al., 2008; Rune et al., 2013), whereas such mice have an impaired glucose tolerance

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later in life (Rune et al., 2013). Various antibiotic cocktails have been applied for these kind of studies (Hansen et al., 2015). However, no matter which other components are used in such antibiotic cocktails, ampicillin seems to be the key component responsible for the major part of the effect of all antibiotic cocktails used for this purpose (Ubeda et al., 2010). Ampicillin belongs to the penicillin group of beta-lactam antibiotics and is able to penetrate both Gram-positive and Gram-negative bacteria. Although new findings have partly elucidated to which extend ampicillin and other antibiotic mixtures eradicate various GM species (Schubert et al., 2015), it is unclear to which extent it suppresses or eradicates the gut bacteria,

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and to which extent environmental bacteria help the entire bacterial community to recover after ampicillin treatment. Studies have previously described alteration and recovery of the GM in human subjects after ampicillin treatment with the majority of subjects returning to their original state after 30 days (De La Cochetière et al., 2005). Also mouse studies have previously shown that ampicillin fails to eradicate the GM, but these studies either used a dose lower (0.5 g

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l-1 drinking water) (Schubert et al., 2015) than commonly applied for full experimental GM

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eradication (1 g l-1 drinking water) (Hansen et al., 2015) or a GM characterization method of lower resolution (Pang et al., 2012). If a complete GM does not recover, the interpretation of

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results from temporary microbiota depletion, and the idea that the GM can be turned on and off,

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becomes questionable.

As such, there is a strong need within this type of animal experimentation to get further

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information on, (i) which members of the GM are eradicated or rather only suppressed by ampicillin treatment, (ii) to which extent these bacteria have the potential to re-establish

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themselves after antibiotic treatment, and (iii) whether the response in the host reflects a temporary depletion of bacteria or rather a permanent change in bacterial stimulation. We,

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therefore, hypothesized that ampicillin treatment would only suppress and not eliminate all bacterial species, that suppressed bacteria would re-appear in the gut even if the mice were kept isolated from environmental bacteria, and that the GM composition of ampicillin treated mice

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after treatment would differ from that of untreated control mice even after being exposed to environmental bacteria.

Materials and Methods Mice

Mothers of the strains BALB/cJBomTac (BALB/c) and C57BL/6JBomTac (B6), both from Taconic Ltd., (Ll. Skensved, Denmark) were housed in open macrolon type III cages (Tecniplast, Varese, Italy) without filter lid in the AAALAC accredited barrier-protected facility

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at the Frederiksberg Campus of the Faculty of Health and Medical Sciences (SUND) at University of Copenhagen. Mice were maintained with aspen bedding nestlets, houses and gnawing sticks (Tapvei, Estonia) under a 12-hour light-dark cycle (6 AM to 6 PM) with a 22 ± 2°C room temperature and a 50-60% relative humidity. Cages and water bottles were changed once weekly (bi-weekly during ampicillin treatment). All animals were fed a standardized

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Altromin 1324 chow diet (Brogaarden A/S, Gentofte, Denmark) and standard quality tap water

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ad libitum. The Animal Experiments Inspectorate under the Ministry of Environment and Food approved the study according to the principles of the European Union Directive 2010/63/EU

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and the Danish Animal Experimentation Act (No 474 15/05/2014).

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Treatments and collection of samples

Ampicillin (Ampivet vet. 7.5 g, Boehringer Ingelheim, Copenhagen, Denmark) was

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administered in sterile ad libitum drinking water with a concentration of 1 g l-1 . For isolation a plastic film isolator (IsoTech, Montreal, Canada) was used and sterilized with Clidox-S

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(Pharmacal Ltd., Waterbury, CT) (Figure 1). Samples collected from the isolator were taken weekly for three weeks prior to use and cultivated on brain-heart infusion agar plates (SSI

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Diagnostics, Copenhagen, Denmark), both aerobically and anaerobically at 37C for 48 h, to confirm germ-free conditions. All animals for this study part were given irradiated bedding

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material and diet as well as sterile water throughout the study. Collected faecal samples were immediately frozen in liquid nitrogen and stored at -80C until analysis. After euthanasia, the colon length was measured and the caecum was weighed. Mesenteric lymph nodes (MLN) and 5-6 ileal Peyer’s patches (PP) were placed in ice-cold phosphate buffered saline (PBS) until sample processing, and 5-10 cm of ileum emptied from intestinal content were removed and snap-frozen on dry-ice and stored at -80◦ C. Experimental setup

Pups used in this study were weaned at three weeks of age, separated according to mouse strains (BALB/c and B6), randomized into three experimental groups with approximately the same

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number of males and females (58 males/57 females) and 2-3 mice per cage (Figure 1). For two of the experimental groups within each mouse strain ampicillin was administered in sterile drinking water. One group of each strain was used as untreated control supplied only with sterile water. After two weeks of ampicillin administration mice were put back on the sterile drinking water, and one ampicillin treated group of each mouse strain was moved from the

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barrier facility to a sterilized plastic isolator. The two remaining groups were conventionally

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housed during re-colonisation (Figure 1). Faecal samples were collected for analysis of GM composition, prior to the ampicillin administration (three weeks old animals, baseline), directly

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after the ampicillin administration (five weeks old animals), and four weeks after re-

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colonisation (nine weeks old animals) (Figure 1). At the age of nine weeks, animals were weighed and euthanized by cervical dislocation. The colon length was measured and the caecum

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was weighed. Immunological characterizations were carried out in a separate group of BALB/c mice and performed in MLN, 5-6 ileal PP, and emptied ileum using two groups of 28 and 27 mice, which were however, only treated with ampicillin for one-week post weaning, and

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terminated three weeks post treatment (Figure 1). At the end of the treatment, half of the

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animals were terminated and samples were collected for analysis. The remaining animals were housed (in conventional conditions) for another three weeks without antibiotics with weekly weighing before sampling.

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16S rRNA gene amplicon high-throughput sequencing

DNA of faecal samples was extracted (Castro-Mejía et al., 2016) and sequencing libraries were built with amplicons derived from the V3-V4 region of the 16S rRNA gene as previously lined out (Jørgensen et al., 2014) (31 cycles within the 1st PCR round [amplification] and 14 cycles for the 2nd PCR round [barcoding] using the Nextera Index Kit®, Illumina). Pair-ended amplicon reads (with corresponding quality scores) were trimmed and merged (>97% quality) using the CLC Genomic Workbench 7.0.4 (CLC bio, Århus, Denmark). Operational taxonomic units (OTUs) clustering (97%) and removing of chimeric sequences was performed using

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UPARSE (Edgar, 2013), and taxonomic assignments were performed using the GreenGenes database (version 12.10) (McDonald et al., 2012). All sequenced samples have been made available through the European Nucleotide Archive (ENA) under the accession number [ENA: PRJEB2015]. Immunological characterization

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Flow cytometry was performed on Peyer’s Patches (PP) and mesenteric lymph nodes (MLN) on

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the day of sampling. PP samples were mashed in ice-cold phosphate buffered saline and filtered

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with a 70 μm filter prior to plating and analysed on a BD Accuri C6 (BD Biosciences, New Jersey, U.S.A.) using the antibody clones/dyes 17A2/FITC for CD3, U5A2-13/PE for NK-NKT,

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53-6.7/PerCP-Cy5.5 for CD8a, GK1.5/APC for CD4, FJK-16S/PE for FoxP3, and RM45/PerCP-Cy5.5 for CD4 (eBioscience Inc., San Diego, CA). The recorded data/events were

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filtered/gated using BD Accuri C6 Software version 1.0.264.15 (BD Biosciences, New Jersey, NY) using a standardized gating strategy described in the figure legend.

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Ileum samples were homogenized in 300 μl of MSD Tris Lysis Buffer containing protease and phosphatase inhibitors (Meso Scale Discovery, Rockville, MD) using a POLYTRON PT 1200 E

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Manual Disperser (Kinematica, Lucerne, Switzerland), and stored at 5°C for 20-30 minutes before centrifugation at 7.500 g for 5 minutes at 5°C. The supernatant was transferred to

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Eppendorf tubes, and stored at -80°C until analysis using the MSD MULTI-SPOT Assay System, specifically a V-PLEX Proinflammatory Panel 1 (mouse) Kit (Meso Scale Discovery) to analyse the following cytokines: IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, CXCL1, IL-10, IL12p70, and TNF-α following the manufacturer’s guide for preparation of samples. Reading of plates was performed on a MESO QuickPlex SQ 120 (Meso Scale Discovery). Readings were imported into MSD Discovery Workbench (Meso Scale Discovery), and cytokine concentrations were calculated based upon a standard curve.

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Statistics

Final body weight, colon length, and caecum weight were compared in GraphPad Prism version 6 and 7 (GraphPad Software, La Jolla, CA) in a one-way ANOVA on a two-tailed hypothesis applying D'Agostino & Pearson omnibus normality test and Brown-Forsythe’s test for equal variances. Statistical analyses of immunological data were done in Minitab version 17 (Minitab

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Inc, Coventry, United Kingdom). For data not displaying a normal distribution in Anderson-

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Darling’s test or equal variances in Levene’s test, analyses were performed on log10 transformed or ranked data. A general linear model multifactorial ANOVA with ampicillin treatment versus

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control as primary readout, sex as secondary readout and time point as tertiary readout was applied on a two-tailed hypothesis. Student’s t-test was used to test differences between

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treatment groups at specific time points, if the overall model showed a change in cell numbers from the sampling directly after ampicillin treatment to the sampling three weeks later. When

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both sex and time had a significant influence on the model Tukey’s post hoc comparison of groups was used to correct for multiple comparisons. p <0.05 was considered significant. High-

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throughput sequencing data were analysed with QIIME (version 1.7 and 1.8) (Caporaso et al., 2010), using a subsampled dataset with 90% of the sequences contained within the most

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indigent sample. Beta-diversities (on weighted UniFrac distances) were evaluated with Adonis (test 999 permutations). The prevalence of operational taxonomic units (OTUs) associated to a

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given mouse group was evaluated with the g-test of independence, while differences in taxa abundance were tested with one-way ANOVA (paired-comparison), for both tests Bonferroni correction was applied (adjusted p-value). For mean OTUs diversity (alpha-diversity) was determined as a function of sequence depth, whereas differences in the number of observed OTUs were calculated using non-parametric Monte Carlo test.

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Results Caecum remained enlarged in ampicillin-treated mice housed in isolators while body weight was not affected

For both BALB/c and B6 strains, caeca were significantly heavier after treatment in both groups

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treated with ampicillin compared to the untreated mice (p <0.0001, Figure 2A-B). There was no significant difference in caecum weight between sexes. The colons of untreated BALB/c mice

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were significantly longer compared to the ampicillin treated mice housed conventionally and in

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isolators (p <0.01 and p =0.05 respectively, Figure 2), but in contrast the colons of untreated B6 mice were significantly shorter compared to the ampicillin treated isolator housed mice (p

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<0.001, Figure 2). The opposing result was particularly due to a large difference in colon length between the strains of untreated mice, whereas this was not evident to the same degree in the

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ampicillin treated groups. There were no significant differences in the final body weights due to ampicillin treatment in both BALB/c and B6 mice (Figure 2E-F). For both mouse strains, male

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mice were significantly heavier than females (p <0.001). Different GM members appeared after ampicillin treatment in conventionally compared to

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isolator housed mice

Sequencing of the 16S rRNA gene (V3-V4 region) yielded 8,310,047 high quality reads (mean

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sequence length of 387 bp) and the number of reads per sequenced sample varied from 18,380 to 200,581 with an average of 75,545 (SD 47,792). The initial GM screening prior to administration of ampicillin showed no differences between experimental groups and mouse strains (Figure 3A-B). No 16S rRNA gene amplicon-sequences were obtained for screening of GM diversity on antibiotic-treated mice directly after treatment, which was interpreted as a low number of bacterial cells (and reduced copies of the 16S rRNA gene) remaining in the gastrointestinal tract as earlier reported (Reikvam et al., 2011).

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In the post-treatment period, there was a profound influence of the environment on the community structure based GM composition profile of both ampicillin-treated groups compared with the untreated mice (Figure 3C). Likewise, significant changes in alpha diversity were found (p <0.01)indicating that in none of the ampicillin treated groups a full recovery took place (Figure 3D). The relative bacterial abundance of the untreated mice was dominated by three

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unclassified genera belonging to the Clostridiales, Rikenellaceae, and S24-7 families, which

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altogether constituted on average 72% of the total bacterial community (Table 1). In both of the ampicillin-treated groups the distribution of the aforementioned taxa comprised less than 1% of

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the total community after the re-colonisation period (Table 1). Moreover, it made a major

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difference for the GM of the mice whether they were isolated or not from the environment (Figure 3). In the conventionally housed ampicillin-treated mice Bacteroides and an unclassified

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genus representing the Lachnospiraceae family accounted for over 95% (Table 1). In the ampicillin-treated mice under isolated conditions, the GM was largely dominated by Blautia and two other GM members assigned to Lachnospiraceae and Enterobacteriaceae families that

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together constituted 86% of the relative abundance (Table 1). Additionally, g-test analysis (p

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<0.05) revealed that the prevalences of Bilophila and Akkermansia were associated with the conventionally housed ampicillin-treated mice compared to isolator housed ampicillin-treated mice (Table 1).

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Ampicillin treatment reduced the innate immunity and regulatory T-cells, and up-regulated the natural killer T-cells in the gut

Ampicillin treatment did not alter the overall percentages of CD4+ cells, i.e. the total fraction of Ths and Tregs (Figure 4A-B), but reduced the population of FoxP3+ cells, i.e. the Tregs, in the MLN (p <0.01; Figure 4D); an effect that was no longer observed three weeks after termination of the treatment. In the PP, the proportion of cytotoxic CD8+ T-cells was substantially larger in the ampicillin-treated mice compared to the untreated mice (p =0.06; Figure 4E), which was also a non-permanent effect. In contrast, the NKT cells were not affected during ampicillin

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treatment, but were significantly increased in the PP three weeks after ampicillin treatment compared to untreated mice (p <0.01; Figure 4G). There were no significant differences in cytokine levels between the two time points during and after ampicillin treatment, however, decreased levels of IL-1β, TNF-α, CXCL1, IL-12 and IL-6 were observed in ileum of ampicillin

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treated mice compared to untreated mice (p <0.05; Figure 5).

Discussion

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Ampicillin in a dose of 1g l-1 drinking water induced a state, at which the number of 16S rRNA

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gene copies dropped below detection level. In line with previous findings, our data demonstrate that antibiotic treatment had a profound effect on the caecum appearance and overall GM

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structure (Reikvam et al., 2011). The mice appeared with a macroscopically “germ-free-like” mouse phenotype as long as four weeks after treatment, with reduced bacterial richness and

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shifted relative distribution, and with caeca of treated mice being significantly heavier compared to untreated mice. Enlarged caeca are one of the main macroscopic characteristics of germ-free

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animals (Bleich and Hansen, 2012), and we have also previously reported that caecum in mice during ampicillin treatment increases to a size fully comparable to those of germ-free mice

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(Ellekilde et al., 2014). Yet, this is the first time that we can describe that such a state is conserved four weeks after treatment. Although this may at the first glance appear to be an

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improvement compared to a previous study, in which ampicillin in a lower dose did not induce a completely germ-free state (Schubert et al., 2015), it is quite clear that this was also not the case in our study, as Blautia spp., Lachnospiraceae, and Enterobacteriaceae although being suppressed sufficiently during the treatment to drop below the detection limit seemed to propagate well in isolator housed mice, even in the presence of an enlarged caecum. Blautia spp. and Lachnospiraceae correlate with low levels of colonic inflammation, as they are good polysaccharide degraders (Eren et al., 2015), and important producers of short chain fatty acids (Zhang et al., 2009), while Enterobacteriaceae are generally pro-inflammatory (Lupp et al., 2007). It was clear that four weeks after treatment the GM of ampicillin treated mice was not

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restored to a composition comparable with the untreated control mice, as there was a more than tenfold reduction in the number of bacterial species and a change in the overall bacterial distribution compared to the untreated control mice. Interestingly, OTUs assigned to the Bacteroides genus propagated dramatically well in conventionally housed animals and to a higher extent than in untreated mice, possibly because they are bacteria primarily caught from

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the environment, which propagate well with no competing species present (in addition to the

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background distribution observed in isolated conditions 0.06%, Table 1). It may seem counterintuitive that mice placed in isolators would experience a post-treatment period with

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diversity more or less comparable to mice placed in the conventional room, where the likelihood

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of contact with new environmental microbes would presumably be higher, but it can be speculated that increasing distribution of specific Bacteroides spp. might reduce distribution of

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Lachnospiraceae and Enterobacteriaceae by exerting detrimental interactions. For example, it has been documented that isobutyrate-producing B. fragilis can inhibit C. perfringens growth and sporulation (Wrigley, 2004). The effect of Bacteroides spp. on promoting health and

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disease seems to be quite diverse and species-specific dependent as reported in numerous

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studies dealing with several immunological diseases (Gauffin Cano et al., 2012; Hsiao et al., 2013; Kim et al., 2005; Mazmanian et al., 2008; Nakano et al., 2006; Wu et al., 2009). Similarly, the properties of the environmentally obtained microbiota can also be very diverse

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and therefore play either beneficial or detrimental roles within the gut. These alterations associated with GM composition can persist for several weeks (Croswell et al., 2009; Schubert et al., 2015), just as observed in our experimental setup. In turn, this could open opportunities to outcompete host-derived bacteria and therefore increase the susceptibility for acquiring a foreign and different microbiota (Croswell et al., 2009; Schubert et al., 2015), which as we report here, is specially influenced by the environment as a restoration driver of the GM composition.

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It is not surprising that ampicillin had the ability to change the immune response when the GM was severely suppressed, but the long-lasting immune-modulatory effect of a temporary treatment period is challenging to interpret due to the significant shift in GM composition post treatment. It has previously been found that both GM and an LPS-rich diet drive the expansion of CD4+FoxP3+ regulatory T-cells in MLN without affecting the proportion of these cells in

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other lymphoid organs, when conventional mice are compared to truly germ-free mice (Hrncir

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et al., 2008). Although we also observed that the population of FoxP3+ regulatory T-cells decreased in the MLN after ampicillin treatment, this effect was not long lasting, which is likely

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to be due to the fact that in our study the mice were not germ-free anymore at this time point.

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This only temporary effect on regulatory T-cells is in contrast to what we previously reported in germ-free mice recolonized at an early age (Hansen et al., 2012). The NKT cells were

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significantly increased in the PP three weeks after ampicillin treatment compared to mice that had not been treated with antibiotics early in life, and it is, therefore, likely that the Bacteroides dominating the GM post treatment restored the population of regulatory T-cells and was

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responsible for the high abundant NKT cell subset in the ampicillin treated group. Bacteroides

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is in fact a known inducer of regulatory T-cells (Round and Mazmanian, 2010) and can produce alpha galactosyl-C18-ceramide, which promotes natural killer T cell proliferation (Kawano et al., 1997). In contrast, ampicillin treatment also decreased the levels of IL-1β, TNF-α, CXCL1,

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and IL-12 in ileum, but time did not seem to be a normalizing factor for any of these. The longlasting effect could be attributed to permanent effects of an early ‘germ-free’ state, which is often the purpose of inducing such a temporary antibiotic treatment, or be due to the reduced GM diversity post treatment, which likely was not sufficient to restore cytokine levels in the gut. Another study using the popular cocktail of ampicillin, vancomycin, neomycin, and metronidazole found that most antibiotic-induced alterations in the gut can be explained by three factors: depletion of the microbiota, direct effects of antibiotics on host tissues and the effects of remaining antibiotic-resistant microbes (Morgun et al., 2015). Down-regulation of different aspects of immunity is mostly related to GM depletion, but as the two other factors

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generate additional impacts, this also adds to the picture that the overall phenotype of antibiotic treated mice differ from that of germ-free mice (Morgun et al., 2015).

Conclusions Temporary ampicillin treatment suppresses the majority of the GM members, but not all of

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these are permanently eliminated. The GM composition is re-established differently as to whether the treated mice are subsequently housed in a microbe containing environment or not,

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but the treated mice do not re-obtain a complex GM comparable to untreated mice regardless of

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housing conditions. Thus, the implications and interpretations for disease development in studies using antibiotics to induce a temporary ‘germ-free’ state can be problematic as GM

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recovery is highly dependent on the environmental surroundings. It should be studied, whether it might be a better solution to re-inoculate the mice with a pre-treatment microbiota or co-house

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treated mice with untreated mice post treatment to reassure a complete re-colonisation process.

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Acknowledgments This work was supported with grants from University of Copenhagen, Excellence Programme for Interdisciplinary Research project “CALM” (JLC), Villum Foundation (MJ), the Danish Strategic Research Council project “Neomune” (ŁK), and the Novo Nordisk & University of Copenhagen Centre ‘LifePharm’ (KMB). The funding sources had no role in study design, data

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collection and interpretation, or the decision to submit the work for publication.

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Figure legends Figure 1 Experimental design for testing the impact of ampicillin treatment and recovery on gut microbiota in BALB/cJBomTac (BALB/c) and C57BL/6JBomTac (B6) mice, as well as

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the subsequent immune response in BALB/cJBomTac (BALB/c) mice.

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Cecum weight (A-B), colon length (C-D), and body weight (E-F) of BALB/cJBomTac (left column) and C57BL/6JBomTac (right column) mice which were kept untreated or

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subjected to ampicillin treatment for two weeks and subsequently housed untreated

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either conventionally (CONV) or in isolators (ISO) for four weeks. Means are shown. ** indicate p < 0.01.; *** indicate p < 0.001; **** indicate p < 0.0001.

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PCoA plots displaying GM beta-diversity (weighted UniFrac) of faecal samples at baseline (A-B), between experimental groups (A) and mouse strains (B), as well as post-treatment period between the same experimental groups (C). Plots are based on

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distance metrics generated by the Jackknifed Beta Diversity workflow calculated using 16,542 reads per sample. Adonis test was calculated using 1,000 permutations. Alphadiversity boxplot depicts the number of observed OTUs among the experimental groups at the post-treatment period (D). Differences in alpha-diversity were based on 16,502 reads per sample and analysed through non-parametric Monte Carlo test (permutations 1,000).

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Figure 4 Flow cytometric analysis of cells isolated from Peyer’s patches (left column) and mesenteric lymph nodes (right column) in untreated control mice (CON) and ampicillin treated mice (AMP) right after treatment (DAY 0) and three weeks after ampicillin

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treatment (DAY 21). Percentages of CD4 positive cells out of the CD3 positive population (T helper cells; A-B), FoxP3 positive cells out of the CD4 positive

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population (regulatory T cells; C-D), CD8 positive cells out of the CD3 positive

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population (cytotoxic T cells; E-F), and NKT positive cells out of the CD3 positive population (natural killer T cells; G-H) are shown. There was no detectable effect of

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sex. Means are shown. ** indicate p < 0.01.

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Figure 5

Protein levels of IL-1β (A), TNF-α (B), CXCL1 (C), IL-12p70 (D), IL-6 (E), IL-10 (F),

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IL-4 (G), IL-5 (H), IFN-γ (I), and IL-2 (J) were measured using a multiplex assay on

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homogenized ileum tissue sampled from ampicillin treated and untreated control mice right after treatment and three weeks after ampicillin treatment. Means are shown for data pooled between the two time points within each experimental group, as time was

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not a dependent variable in the two-way ANOVA performed. * indicates p < 0.05 in two-way ANOVA.

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Table 1. Relative abundance of bacterial genera determined in faecal samples of mice (BALB/c and B6) subjected to ampicillin treatment for two weeks and subsequently housed in either isolator (Amp–Iso) or conventionally housed (Amp–Conv) for four weeks. Controls were untreated throughout the period.

Bacilli

YS2 Lactobacillal es

unclassified Enterococcace ae

Firmicutes Firmicutes

Clostridia

Clostridiales

Firmicutes

Clostridia

Clostridiales

Firmicutes

Clostridia

Clostridiales

Firmicutes

Clostridia

Clostridiales

Firmicutes

Clostridia

Clostridiales

Firmicutes

Clostridia

Clostridiales

Firmicutes

Clostridia

Clostridiales

unclassified Lachnospirace ae Lachnospirace ae Lachnospirace ae Lachnospirace ae Peptostreptoco ccaceae Ruminococcac eae

Firmicutes Proteobact eria Proteobact eria Proteobact eria Verrucomi crobia Unassigne d

Clostridia Deltaproteoba cteria Gammaproteo bacteria Gammaproteo bacteria Verrucomicro biae

Clostridiales Desulfovibri onales Enterobacteri ales Enterobacteri ales Verrucomicr obiales

Other Desulfovibrion aceae Enterobacteria ceae Enterobacteria ceae Verrucomicrob iaceae

Bacteroidia Bacteroidia Bacteroidia Bacteroidia Bacteroidia

Prevotellaceae Rikenellaceae S24-7 Other

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Bacteroidaceae

Genus Bacteroide s Prevotella unclassifie d unclassifie d

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Order Bacteroidale s Bacteroidale s Bacteroidale s Bacteroidale s Bacteroidale s

Other unclassifie d Enterococ cus unclassifie d unclassifie d

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Phylum Bacteroidet es Bacteroidet es Bacteroidet es Bacteroidet es Bacteroidet es Cyanobact eria

Mean values with different superscripts (

a, b, c

M ouse groups AmpControls Conv 6.68 89.2 b % 5% 1.49 <0.0 a % 0% 20.3 0.02 a 7% % 28.7 0.05 a 9% % 0.08 <0.0 a % 0% 0.30 a % <0.0 0.01 b 0% % 23.6 0.46 a 2% % 4.57 6.28 b % % 0.03 0.95 b % % 0.40 <0.0 a % 0% 0.30 0.06 b % % <0.0 0.16 b 0% % 1.81 a % 0.14 <0.0 a % 0% 0.11 % 0.02 0.39 b % % <0.0 0% <0.0 2.01 b 0% % 0.76 0.03 a % %

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Taxa

Blautia [Ruminoco ccus] Other unclassifie d Oscillospir a Other Bilophila unclassifie d Erwinia Akkermans ia

a

b

b

b

b

b

Amp-Iso 0.06 b % <0.0 b 0% 0.01 b % 0.01 b % <0.0 b 0% <0.0 b 0% 0.05 a %

b

b

b

47.3 0% 21.2 2%

a

b

b

b

b

a

b

b

a

b

1.24 % 1.48 % 0.08 % 0.01 % <0.0 0% 17.9 2% 0.03 % <0.0 0% 0.03 %

) were significantly different in multiple

comparison through one-way ANOVA (paired-comparison using 1,000 rarefied OTU tables and 16,542 sequences per sample). All probabilities were adjusted with the Bonferroni correction.

a

a

a

b

b

b

a

a

b

b

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Graphical abstract

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Highlights

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Ampicillin treatment induces an apparently germ-free status of mice A microbiota will re-colonize even in mice isolated in germ-free surroundings Post-treatment microbiota composition is highly dependent on the environment Post-treatment microbiota is significantly different from non-treated mice The immune system of treated mice differs from non-treated mice after treatment

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