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The effect of having Christmas dinner with in-laws on gut microbiota composition
Human Microbiome Journal 13 (2019) 100058
Contents lists available at ScienceDirect
Human Microbiome Journal journal homepage: www.elsevier.com/locate/humic
Original Article
The effect of having Christmas dinner with in-laws on gut microbiota composition
T
⁎
Nicolien C. de Clercqa, , Myrthe N. Frissena, Evgeni Levina,b, Mark Davidsa, Jorn Hartmana, Andrei Prodana,b, Hilde Herremaa, Albert K. Groena,c, Johannes A. Romijna, Max Nieuwdorpd,e a
Department of Internal and Vascular Medicine, Amsterdam University Medical Centre, Location AMC, 1105 AZ Amsterdam, the Netherlands Horizon BV, 3062 ME Rotterdam, the Netherlands c Department of Pediatrics, University of Groningen, University Medical Center Groningen, 9713 GZ Groningen, the Netherlands d Department of Internal and Vascular Medicine, Amsterdam University Medical Centre, Location AMC and VUMC, 1105 AZ Amsterdam, the Netherlands e Wallenberg Laboratory, University of Gothenburg, Gothenburg, Sweden b
A R T I C LE I N FO
A B S T R A C T
Keywords: Gut microbiota Gut-brain-axis In-laws Stress Christmas
The Christmas season can have a major impact on human health. Especially increased contact with in-laws during the holiday season is an important environmental factor known to affect both physical and mental health (Mirza et al., 2004). However, the mechanism through which in-laws influence host health is not yet understood. Emerging evidence has identified the intestinal microbiota as an important mediator for both physical and mental health. Here, we performed a prospective observational study to examine the impact of contact with inlaws on the gut microbiome during the Christmas season. We conducted 16S ribosomal DNA sequencing of fecal samples collected at two separate time points (December 23rd and December 27th 2016) from a group of 28 healthy volunteers celebrating Christmas. To discriminate between participants who visited their own family versus their in-laws, we built a multivariate statistical model that identified microbial biomarker species. We observed two distinct microbial-biomarker signatures discriminating the participants that visited their in-laws versus their own family over the Christmas season. We identified seven bacterial species whose relative-change profile differed significantly among these two groups. In participants visiting in-laws, there was a significant decrease in all Ruminococcus species, known to be associated with psychological stress and depression. A larger randomized controlled study is needed to reproduce these findings before we can recognize in-laws as a potential risk factor for the gut microbiota composition and subsequently host health.
1. Introduction Christmas and feces appear to have a traditional yet mysterious connection. Take a close look at any nativity scene in Catalonia (Spain) and you will spot Caganer; a porcelain man defecating in the presence of Mary, Joseph, and their newly born son (Fig. 1). This defecating man entered the scene around the 18th century and is said to symbolise fertilisation of the holy ground, hoping for a prosperous harvest in the new year [2]. Spain is not exclusive in its association between Christmas and feces. Unknown to many, the term ‘mistletoe’ derives from the Anglo Saxon words ‘mistel’- meaning ‘dung’- and ‘tan’-meaning ‘stick or twig’ [3]. Hence, ‘mistletoe’ can basically be translated into ‘poo on a stick’.
And more recently, let us not forget “Mr. Hankey, the Christmas Poo”, the 1999 South Park hit that peaked at No. 4 on the Official UK Singles Chart [4]. What makes our Christmas poo distinct from any other Number Two’s throughout the year? If we examine poo at a more detailed level, a complex community of microorganisms is revealed: the gut microbiota. The notion is that these gut microbiota function as a metabolic ‘organ’ involved in health and disease [5]. Environmental factors, such as altered diet, intoxications (i.e. alcohol) and psychological stress, disturb the core composition of the gut microbiota [6], which is associated with pathological conditions such as obesity [7,8], metabolic syndrome [9,10] and inflammatory bowel disease [11]. Identification of the environmental factors that play a role in the modulation of the
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Corresponding author. E-mail addresses: [email protected] (N.C. de Clercq), [email protected] (M.N. Frissen), [email protected] (E. Levin), [email protected] (M. Davids), [email protected] (J. Hartman), [email protected] (A. Prodan), [email protected] (H. Herrema), [email protected] (A.K. Groen), [email protected] (J.A. Romijn), [email protected] (M. Nieuwdorp). https://doi.org/10.1016/j.humic.2019.100058
Available online 03 July 2019 2452-2317/ © 2019 Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
Human Microbiome Journal 13 (2019) 100058
N.C. de Clercq, et al.
activities implicated in managing emotions [18]. Due to extensive differences in the methodology of intervention studies in humans, identifying strain-specific effects is a next step in untangling the interconnectedness of social factors with microbial profiles. The Christmas season serves as a natural intervention of a sudden increase in contact with in-law family members. To determine whether gut health could be implicated in mediating in-law induced health problem, we performed an observational study examining the effect of visiting in-laws during Christmas on the gut microbiota composition. We conducted 16S ribosomal DNA sequencing of fecal samples collected at two separate time points (December 23rd and December 27th 2016) from a subgroup of participants who visited their own family versus their in-laws. A multivariate statistical model was built to identify particular microbial species associated with visiting in-laws or own family members. 2. Methods 2.1. Participants and study design Fig. 1. El Caganer. With permission of Maria Schnabel Communications.
Participants were eligible for inclusion if they were of Caucasian descent, between 20 and 40 years old with a body-mass index (BMI) between 18 and 25 kg/m2, since the gut microbiota composition is partly influenced by age, BMI and ethnical background. Exclusion criteria were defined by factors known to influence gut microbiota composition: adherence to a specific diet (e.g. vegetarian or vegan), smoking or substance abuse, any medication use in the past three months or a history of cholecystectomy. Approval for the implementation of this study was obtained from the AMC ethical committee and informed consent from all participants was obtained. The total study period was seven days during which fecal samples were provided at two time points (T1 on December 23rd and T2 on December 27th) (Fig. 2). Participants were instructed to collect fresh morning fecal samples and transport these to the Research Centre on ice (approximately −4 °C) within six hours after sampling. On site, samples were stored at −80 °C until analysis was performed. Additionally, participants were asked to keep a detailed daily log of their complete dietary intake starting December 21st up until December 26th. Finally, participants reported whether they visited only their own family and/or their inlaws during Christmas.
gut microbiota is of great interest, as these factors may directly and indirectly influence host health. The Christmas season is a period during which multiple environmental changes are prominently present. The effect of altered diet and alcohol on the gut microbiota has already been studied extensively, however during Christmas individuals are exposed to another environmental factor that might have an unexpected influence on the gut microbiota composition: the mandatory visit to in-laws. In the past decade, studies have highlighted the impact of social interactions with in-law family on both physical and mental health. For example, women living together with their in-laws are up to three times more likely to develop coronary heart disease [12]. Mirza and colleagues moreover found that relational problems with in-laws were associated with anxiety and depressive disorders [1]. It is hypothesized that perceived psychosocial stress mediates these effects, but the exact mechanism through which stress affects host health, is still unknown. The human gut microbiota are becoming increasingly known for their link between mental and physical well-being. Not only can psychological stress alter gut microbial composition, manipulation of the microbial signature also affects how stressors are perceived. For instance, rodent studies have shown that stressful interventions induce changes in the gut microbiota composition [13]. While this comes with symptoms of anxiety and depression in regular mice, germ free animals fail to show this outcome when subjected to the same stressors [13]. Vice versa, harmful shifts in microbial patterns are suggested to lead to anxiety-like behavior through causing a pro-inflammatory state triggered by translocation of bacterial components [14]. The relationship appears to be causal, since a restored homeostasis of the intestinal environment, for example through the administration of probiotics or by cohousing, can undo these effects [15]. The bidirectional pathways linking the gut with the brain, and physical with mental well-being, is also referred to as the (microbiota-)gut-brain axis [14]. Evidence from human studies are in line with these findings, with an increased occurrence of bacterial translocation in those suffering from psychiatric disorders like depression [16,17]. Moreover, a probiotic intervention in healthy volunteers showed significant changes in brain
2.2. Fecal analysis Fecal material was lysed using high-SDS lysis buffer (4% (w/v) SDS, 50 mM TrisHCl pH 8, 500 mM NaCl, 50 mM EDTA) and repetitive beadbeating (RBB) [19]. Genomic DNA was isolated and purified using a Maxwell® 16 Instrument (Promega) according to manufacturers’ protocol. DNA quality was assessed on agarose gel; only high-quality DNA was used for further analysis. 16S rRNA gene (V3-V4 region) was amplified using an adapted PCR method [20]. Among the variable regions of 16 s gene V3 and V4 are highly variable regions which can distinguish bacterial subtypes, achieve good domain specificity, high coverage and a broad spectrum in the bacteria domain. PCR product was purified (FX robot) and pooled (equimolar) prior to sequencing analysis using Illumina Miseq (V3, 600) [20,21]. Samples were demultiplexed using only exact barcodes. Data was further processed using dada2 (v1.5.2) [22]). Paired reads were Fig. 2. Timeline of study procedures. Active study days included the 21st–27th of December. On December 23rd and 27th participants provided morning fecal samples ( ). Dietary questionnaires ( ) were filled out from December 21st up and until December 26th.
2
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truncated at position 260 and 210 respectively and ambiguous reads were discarded. Forward and reverse amplicon sequence variants (ASV) were inferred and pairs were merged. Chimeras were removed only if the parent sequence was at least twice as abundant [23]. Taxonomy down to gens level was assigned using the Silva reference database (V128) [24] using RDP [25] and species assignments were performed using exact sequence matching. A phylogenetic tree was generated using R packages phangorn (2.2.0) and DECIPHER (V2.0 Wright 2016).
Table 1 Baseline characteristics family versus in-laws: dietary and alcohol intake.
Age (yr) Total energy (kcal) Carbohydrates (gr/kcal) Protein (gr/kcal) Fat (gr/kcal) Saturated fat (gr/kcal) Fibre (gr/kcal) Alcohol (gr)
2.3. Statistical analyses Our primary endpoint was to evaluate the variation in gut microbiota diversity and subsequent composition between the group that had visited their own family versus their in-laws during Christmas. This was assessed by both weighted (relative abundance/richness) and unweighted (presence/absence) UniFrac distance, by using permutational multivariate analysis of variance (PERMANOVA, adonis) [26]. The large majority of the diversity metrics (e.g. Shannon, alpha, beta, etc.) are generally applicable for groups of subjects, thus, they do not allow detailed description of the microbial signature on for “individual participant” level. Using state-of-the-art statistical learning algorithm enables to identify potential microbial biomarker species that lead to accurate discrimination among the subjects who visited their family versus their in-laws. To discriminate between participants who visited their own family versus their in-laws, we thus built a multivariate statistical model to identify microbial biomarker species. This model measured the degree of change in microbiota (on species-level) for each subject during Christmas. One key factor that allowed successful identification of the microbial-biomarker signature in this complex, high-dimensional dataset was the application of statistical learning methodology, specifically tailored to analyze collected 16S data. In particular, we have used an adapted version of the elastic net algorithm [27] (with Hinge loss function) for identification of the most relevant microbial biomarkers. Furthermore, our statistical learning approach includes stability selection [28] which reflects the frequency that a particular biomarker was identified in multiple simulations on a re-randomized dataset.
In-laws (n = 16)
Family (n = 8)
p-value
28.8 ± 1.9 0.32 ± 0.36 −0.20 ± 0.14 0.13 ± 0.21 0.11 ± 0.17 0.18 ± 0.27 −0.31 ± 0.21 32.76 ± 13.89
30.7 ± 3.2 0.22 ± 0.24 −0.11 ± 0.12 0.01 ± 0.20 0.06 ± 0.21 0.30 ± 0.29 −0.24 ± 0.21 31.70 ± 14.19
0.14 0.45 0.11 0.20 0.53 0.36 0.51 0.864
Relative change in macronutrients and alcohol consumption. Values are expressed as means ± SD. There was no significant difference between participants visiting family versus in-law group (means) tested with ANOVA.
Fig. 3. Relative change in alpha diversity (Shannon) for participants visiting family versus visiting in-laws.
fecal microbiota alpha-diversity (Shannon-index) compared to participants visiting their own family during Christmas (p < 0.05) (Fig. 3). Furthermore, there was a distinct microbial signature for participants who visited their own family versus participants who visited their in-laws (Fig. 4). This signature comprised seven species with significantly affected relative-change profiles between the two groups (p < 0.05). The microbial signature of participants visiting their own family showed significantly higher variations compared to the in-laws. Furthermore, the biomarker signatures were dominated by the family Ruminococcaceae, showing a significant difference in change in three genera. The genus Rumminococcaceae_UCG-009 exhibited the most divergent outcome: an increase in the family group versus a decrease in the in-laws group. The relative change of two other Ruminococcaceae species (Ruminococcaceae_UCG-002 and Ruminococcaceae_NK4A214_group), was decreased in both groups.
3. Results 3.1. Baseline characteristics We recruited 28 healthy Caucasian participants (14 men, 14 women). Four participants were excluded from the analysis. Two participants did not provide fecal samples on the second time point (T2) due to the “yuck-factor” (a feeling of horror or disgust towards procedures involving poo) [29,30]. One participant did not complete the dietary questionnaires. One participant followed an extraordinary dietary regime and fasted the day before Christmas (December 23rd), with a total intake of only 3 kcal. Of the remaining twenty-four participants that could be included in subsequent analyses, sixteen participants visited their in-laws during Christmas and the remaining eight celebrated Christmas with their own family (Table 1). The relative changes in dietary macronutrients and alcohol consumption during Christmas did not significantly differ between those who visited in-laws versus their own family. Both groups reported a relative increase in saturated fats and proteins, due to a greater consumption of animal based products during Christmas (Table 1).
4. Discussion The multivariate statistical model revealed two distinct biomarker signatures containing seven species that are able to distinguish the participants visiting their family from participants visiting their in-laws during Christmas (Fig. 4). We identified three Ruminococcaceae genera with a different degree of change in both groups. In this regard, the genus Rumminococcaceae_UCG-009 exhibited most divergent outcome with a relative increase in the family group versus a relative decrease in the inlaws group. Strikingly, humans with major depressive disorders [31] and mice exposed to chronic stress [32] are found to have significantly lower levers of Ruminococcaceae genes. These findings could imply that visiting in-laws during Christmas induces high stress levels, which in turn reduces the presence of Ruminococcaceae genus in the gut. We did
3.2. Alpha-diversity and microbial signature Amplicon sequences were processed using dada2 yielding a total of 2354 unique amplicon sequence variants with a minimal sample coverage of 53,340 sequences (Additional file 1). Participants visiting their in-laws had a significant higher change in 3
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Fig. 4. Biomarker signatures for participants visiting family versus visiting in-laws. AUC = 0.78 for the difference between family and in-laws. Value between ‘0’ and ‘1’ reflects a decrease in amplicon sequence variants (ASV), value above ‘1’ reflects an increase.
not have participants report on perceived stress in in-law situations, and it is unlikely that all experienced this feeling of distress. Yet finding a significant decrease in the whole-group analysis could imply an underestimation of the effect size. In the subgroup visiting their own family, we also saw a distinct decrease in two different Ruminococcaceae genes. Potentially not only participants visiting their in-laws, but also participants visiting their family experienced increased psychological stress during Christmas. Furthermore, the model showed higher variations in microbial signature for those participants visiting family during Christmas, versus the group who celebrated Christmas with their in-laws. We know from both animal and human studies that social and physical interactions between individuals promotes both species richness (abundance) and microbial diversity, resulting in a so called pan-microbiome [33,34]. We hypothesise that participants visiting their own family had more physical contact with their relatives compared to the in-law group. which might explain the greater variations in microbial species in the family group. These findings could potentially have clinical relevance, since we know that a higher diversity is one of the most important predictors of host health [35].
supervision from the research-team. Despite the standardized questionnaires, the embarrassing and confronting nature of facing one’s food intake, especially during Christmas, and the inconvenience of filling out a dietary assessment are likely to have influenced the reporting [36]. Differences in the completeness of the forms were noted upon analysis and could therefore have attributed to bias in the results.
4.1. Limitations
Acknowledgments
This study was limited by its observational nature. Participants were free of choice whether to visit family or in-laws during Christmas; this decision was not assigned by the investigators. It is possible that participants with a greater aversion towards their in-laws have used the excuse of participating in a scientific study addressing Christmas poo to not visit their in-laws during Christmas. Since we did not score the level of (experienced) stress during Christmas visits, we can only postulate that the difference in biomarker signature between visiting family and in-laws was due to stress. In addition, multiple (un)known confounders might have influenced our main outcome measures. Beyond known factors like diet, gender, BMI, age, physical exercise, pets and individual microbiome variations, there are many other yet to be identified factors that may have influenced our outcomes. Moreover, this study relied on a small group of volunteers as participants, and analysis of a greater pool of individuals would help to identify trends in microbiota changes with greater accuracy. Finally, in this study participants had to report their food intake, without
We would like to thank all study participants, Han Levels and Maaike Winkelmeijer for fecal DNA extraction, Luuk Scheres, Thijs van Mens, Kristien Bouter, Annefleur Koopen, Jurre Olivier and Robert-Jan de Muinck Keizer for their contribution during the scientific discussions regarding the manuscript.
5. Conclusion This study identified a microbial signature that can discriminate between participants who visited their own family versus their in-laws during Christmas. However, observed differences between the potentially stressful event of visiting in-laws during Christmas and changes in the gut microbiota composition are modest. Before we can recognize inlaws as a new environmental factor that can influence the gut microbiota composition and subsequently host health, a larger randomized controlled study is needed to reproduce these findings and confirm a direct link between visiting in-laws, stress and the gut microbiota composition.
Funding This study was sponsored by the Department of Vascular Medicine of the Academic Medical Center of Amsterdam.
Availability of data and materials The raw sequence data set has been deposited in the European Nucleotide Archive under project PRJEB22340. 4
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Contributors
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NC, MF, HH, AK, JR and MN conceived and designed the project. NC and MN collected samples and performed the experiments. NC, MF, EL, MD, AP, HH, JH and AK analyzed and interpreted the data. NC, MF, EL, MD, JH, HH and MN wrote the paper. All authors commented on the manuscript. All authors read and approved the final manuscript. Ethical approval This study was approved by the Medical Ethical Committee of the Academic Medical Center in Amsterdam, The Netherlands. Consent for publications Consent for publication has been obtained from all participants. Declaration of Competing Interest All authors declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work. References [1] Mirza I, Jenkins R. Risk factors, prevalence, and treatment of anxiety and depressive disorders in Pakistan: systematic review. BMJ 2004;328:794. https://doi.org/10. 1136/bmj.328.7443.794. [2] https://en.wikipedia.org/wiki/Caganer. Extr May 2018. [3] http://www.todayifoundout.com/index.php/2010/12/the-word-mistletoe-literallymeans-dung-twig/. No Title. Extr August 1st 2017. [4] Parker T, Stone M. South Park. The Complete First Season: ‘Mr. Hankey, the Christmas Poo’. 272; 2003. [5] Bouter KE, van Raalte DH, Groen AK, et al. Role of the gut microbiome in the pathogenesis of obesity and obesity-related metabolic dysfunction. Gastroenterology 2017;152:1671–8. https://doi.org/10.1053/j.gastro.2016.12. 048. [6] Gill SR, Pop M, DeBoy RT, et al. Metagenomic analysis of the human distal gut microbiome. Science 2006;312:1355–9. https://doi.org/10.1126/science.1124234. [7] Turnbaugh PJ, Ley RE, Mahowald MA, et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006;444:1027–31. https://doi. org/10.1038/nature05414. [8] Bäckhed F, Ding H, Wang T, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 2004;101:15718–23. https://doi. org/10.1073/pnas.0407076101. [9] D’Aversa F, Tortora A, Ianiro G, et al. Gut microbiota and metabolic syndrome. Intern Emerg Med 2013;8:11–5. https://doi.org/10.1007/s11739-013-0916-z. [10] Arora T, Bäckhed F. The gut microbiota and metabolic disease: current understanding and future perspectives. J Intern Med 2016;280:339–49. https://doi.org/ 10.1111/joim.12508. [11] Devkota S, Wang Y, Musch MW, et al. Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice. Nature 2012;487:104–8. https://doi.org/10.1038/nature11225. [12] Ikeda A, Iso H, Kawachi I, et al. Living arrangement and coronary heart disease: the JPHC study. Heart 2008;95:577–83. https://doi.org/10.1136/hrt.2008.149575. [13] Vuong HE, Yano JM, Fung TC, et al. The Microbiome and Host Behavior. Annu Rev
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Contents lists available at ScienceDirect
Human Microbiome Journal journal homepage: www.elsevier.com/locate/humic
Original Article
The effect of having Christmas dinner with in-laws on gut microbiota composition
T
⁎
Nicolien C. de Clercqa, , Myrthe N. Frissena, Evgeni Levina,b, Mark Davidsa, Jorn Hartmana, Andrei Prodana,b, Hilde Herremaa, Albert K. Groena,c, Johannes A. Romijna, Max Nieuwdorpd,e a
Department of Internal and Vascular Medicine, Amsterdam University Medical Centre, Location AMC, 1105 AZ Amsterdam, the Netherlands Horizon BV, 3062 ME Rotterdam, the Netherlands c Department of Pediatrics, University of Groningen, University Medical Center Groningen, 9713 GZ Groningen, the Netherlands d Department of Internal and Vascular Medicine, Amsterdam University Medical Centre, Location AMC and VUMC, 1105 AZ Amsterdam, the Netherlands e Wallenberg Laboratory, University of Gothenburg, Gothenburg, Sweden b
A R T I C LE I N FO
A B S T R A C T
Keywords: Gut microbiota Gut-brain-axis In-laws Stress Christmas
The Christmas season can have a major impact on human health. Especially increased contact with in-laws during the holiday season is an important environmental factor known to affect both physical and mental health (Mirza et al., 2004). However, the mechanism through which in-laws influence host health is not yet understood. Emerging evidence has identified the intestinal microbiota as an important mediator for both physical and mental health. Here, we performed a prospective observational study to examine the impact of contact with inlaws on the gut microbiome during the Christmas season. We conducted 16S ribosomal DNA sequencing of fecal samples collected at two separate time points (December 23rd and December 27th 2016) from a group of 28 healthy volunteers celebrating Christmas. To discriminate between participants who visited their own family versus their in-laws, we built a multivariate statistical model that identified microbial biomarker species. We observed two distinct microbial-biomarker signatures discriminating the participants that visited their in-laws versus their own family over the Christmas season. We identified seven bacterial species whose relative-change profile differed significantly among these two groups. In participants visiting in-laws, there was a significant decrease in all Ruminococcus species, known to be associated with psychological stress and depression. A larger randomized controlled study is needed to reproduce these findings before we can recognize in-laws as a potential risk factor for the gut microbiota composition and subsequently host health.
1. Introduction Christmas and feces appear to have a traditional yet mysterious connection. Take a close look at any nativity scene in Catalonia (Spain) and you will spot Caganer; a porcelain man defecating in the presence of Mary, Joseph, and their newly born son (Fig. 1). This defecating man entered the scene around the 18th century and is said to symbolise fertilisation of the holy ground, hoping for a prosperous harvest in the new year [2]. Spain is not exclusive in its association between Christmas and feces. Unknown to many, the term ‘mistletoe’ derives from the Anglo Saxon words ‘mistel’- meaning ‘dung’- and ‘tan’-meaning ‘stick or twig’ [3]. Hence, ‘mistletoe’ can basically be translated into ‘poo on a stick’.
And more recently, let us not forget “Mr. Hankey, the Christmas Poo”, the 1999 South Park hit that peaked at No. 4 on the Official UK Singles Chart [4]. What makes our Christmas poo distinct from any other Number Two’s throughout the year? If we examine poo at a more detailed level, a complex community of microorganisms is revealed: the gut microbiota. The notion is that these gut microbiota function as a metabolic ‘organ’ involved in health and disease [5]. Environmental factors, such as altered diet, intoxications (i.e. alcohol) and psychological stress, disturb the core composition of the gut microbiota [6], which is associated with pathological conditions such as obesity [7,8], metabolic syndrome [9,10] and inflammatory bowel disease [11]. Identification of the environmental factors that play a role in the modulation of the
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Corresponding author. E-mail addresses: [email protected] (N.C. de Clercq), [email protected] (M.N. Frissen), [email protected] (E. Levin), [email protected] (M. Davids), [email protected] (J. Hartman), [email protected] (A. Prodan), [email protected] (H. Herrema), [email protected] (A.K. Groen), [email protected] (J.A. Romijn), [email protected] (M. Nieuwdorp). https://doi.org/10.1016/j.humic.2019.100058
Available online 03 July 2019 2452-2317/ © 2019 Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
Human Microbiome Journal 13 (2019) 100058
N.C. de Clercq, et al.
activities implicated in managing emotions [18]. Due to extensive differences in the methodology of intervention studies in humans, identifying strain-specific effects is a next step in untangling the interconnectedness of social factors with microbial profiles. The Christmas season serves as a natural intervention of a sudden increase in contact with in-law family members. To determine whether gut health could be implicated in mediating in-law induced health problem, we performed an observational study examining the effect of visiting in-laws during Christmas on the gut microbiota composition. We conducted 16S ribosomal DNA sequencing of fecal samples collected at two separate time points (December 23rd and December 27th 2016) from a subgroup of participants who visited their own family versus their in-laws. A multivariate statistical model was built to identify particular microbial species associated with visiting in-laws or own family members. 2. Methods 2.1. Participants and study design Fig. 1. El Caganer. With permission of Maria Schnabel Communications.
Participants were eligible for inclusion if they were of Caucasian descent, between 20 and 40 years old with a body-mass index (BMI) between 18 and 25 kg/m2, since the gut microbiota composition is partly influenced by age, BMI and ethnical background. Exclusion criteria were defined by factors known to influence gut microbiota composition: adherence to a specific diet (e.g. vegetarian or vegan), smoking or substance abuse, any medication use in the past three months or a history of cholecystectomy. Approval for the implementation of this study was obtained from the AMC ethical committee and informed consent from all participants was obtained. The total study period was seven days during which fecal samples were provided at two time points (T1 on December 23rd and T2 on December 27th) (Fig. 2). Participants were instructed to collect fresh morning fecal samples and transport these to the Research Centre on ice (approximately −4 °C) within six hours after sampling. On site, samples were stored at −80 °C until analysis was performed. Additionally, participants were asked to keep a detailed daily log of their complete dietary intake starting December 21st up until December 26th. Finally, participants reported whether they visited only their own family and/or their inlaws during Christmas.
gut microbiota is of great interest, as these factors may directly and indirectly influence host health. The Christmas season is a period during which multiple environmental changes are prominently present. The effect of altered diet and alcohol on the gut microbiota has already been studied extensively, however during Christmas individuals are exposed to another environmental factor that might have an unexpected influence on the gut microbiota composition: the mandatory visit to in-laws. In the past decade, studies have highlighted the impact of social interactions with in-law family on both physical and mental health. For example, women living together with their in-laws are up to three times more likely to develop coronary heart disease [12]. Mirza and colleagues moreover found that relational problems with in-laws were associated with anxiety and depressive disorders [1]. It is hypothesized that perceived psychosocial stress mediates these effects, but the exact mechanism through which stress affects host health, is still unknown. The human gut microbiota are becoming increasingly known for their link between mental and physical well-being. Not only can psychological stress alter gut microbial composition, manipulation of the microbial signature also affects how stressors are perceived. For instance, rodent studies have shown that stressful interventions induce changes in the gut microbiota composition [13]. While this comes with symptoms of anxiety and depression in regular mice, germ free animals fail to show this outcome when subjected to the same stressors [13]. Vice versa, harmful shifts in microbial patterns are suggested to lead to anxiety-like behavior through causing a pro-inflammatory state triggered by translocation of bacterial components [14]. The relationship appears to be causal, since a restored homeostasis of the intestinal environment, for example through the administration of probiotics or by cohousing, can undo these effects [15]. The bidirectional pathways linking the gut with the brain, and physical with mental well-being, is also referred to as the (microbiota-)gut-brain axis [14]. Evidence from human studies are in line with these findings, with an increased occurrence of bacterial translocation in those suffering from psychiatric disorders like depression [16,17]. Moreover, a probiotic intervention in healthy volunteers showed significant changes in brain
2.2. Fecal analysis Fecal material was lysed using high-SDS lysis buffer (4% (w/v) SDS, 50 mM TrisHCl pH 8, 500 mM NaCl, 50 mM EDTA) and repetitive beadbeating (RBB) [19]. Genomic DNA was isolated and purified using a Maxwell® 16 Instrument (Promega) according to manufacturers’ protocol. DNA quality was assessed on agarose gel; only high-quality DNA was used for further analysis. 16S rRNA gene (V3-V4 region) was amplified using an adapted PCR method [20]. Among the variable regions of 16 s gene V3 and V4 are highly variable regions which can distinguish bacterial subtypes, achieve good domain specificity, high coverage and a broad spectrum in the bacteria domain. PCR product was purified (FX robot) and pooled (equimolar) prior to sequencing analysis using Illumina Miseq (V3, 600) [20,21]. Samples were demultiplexed using only exact barcodes. Data was further processed using dada2 (v1.5.2) [22]). Paired reads were Fig. 2. Timeline of study procedures. Active study days included the 21st–27th of December. On December 23rd and 27th participants provided morning fecal samples ( ). Dietary questionnaires ( ) were filled out from December 21st up and until December 26th.
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truncated at position 260 and 210 respectively and ambiguous reads were discarded. Forward and reverse amplicon sequence variants (ASV) were inferred and pairs were merged. Chimeras were removed only if the parent sequence was at least twice as abundant [23]. Taxonomy down to gens level was assigned using the Silva reference database (V128) [24] using RDP [25] and species assignments were performed using exact sequence matching. A phylogenetic tree was generated using R packages phangorn (2.2.0) and DECIPHER (V2.0 Wright 2016).
Table 1 Baseline characteristics family versus in-laws: dietary and alcohol intake.
Age (yr) Total energy (kcal) Carbohydrates (gr/kcal) Protein (gr/kcal) Fat (gr/kcal) Saturated fat (gr/kcal) Fibre (gr/kcal) Alcohol (gr)
2.3. Statistical analyses Our primary endpoint was to evaluate the variation in gut microbiota diversity and subsequent composition between the group that had visited their own family versus their in-laws during Christmas. This was assessed by both weighted (relative abundance/richness) and unweighted (presence/absence) UniFrac distance, by using permutational multivariate analysis of variance (PERMANOVA, adonis) [26]. The large majority of the diversity metrics (e.g. Shannon, alpha, beta, etc.) are generally applicable for groups of subjects, thus, they do not allow detailed description of the microbial signature on for “individual participant” level. Using state-of-the-art statistical learning algorithm enables to identify potential microbial biomarker species that lead to accurate discrimination among the subjects who visited their family versus their in-laws. To discriminate between participants who visited their own family versus their in-laws, we thus built a multivariate statistical model to identify microbial biomarker species. This model measured the degree of change in microbiota (on species-level) for each subject during Christmas. One key factor that allowed successful identification of the microbial-biomarker signature in this complex, high-dimensional dataset was the application of statistical learning methodology, specifically tailored to analyze collected 16S data. In particular, we have used an adapted version of the elastic net algorithm [27] (with Hinge loss function) for identification of the most relevant microbial biomarkers. Furthermore, our statistical learning approach includes stability selection [28] which reflects the frequency that a particular biomarker was identified in multiple simulations on a re-randomized dataset.
In-laws (n = 16)
Family (n = 8)
p-value
28.8 ± 1.9 0.32 ± 0.36 −0.20 ± 0.14 0.13 ± 0.21 0.11 ± 0.17 0.18 ± 0.27 −0.31 ± 0.21 32.76 ± 13.89
30.7 ± 3.2 0.22 ± 0.24 −0.11 ± 0.12 0.01 ± 0.20 0.06 ± 0.21 0.30 ± 0.29 −0.24 ± 0.21 31.70 ± 14.19
0.14 0.45 0.11 0.20 0.53 0.36 0.51 0.864
Relative change in macronutrients and alcohol consumption. Values are expressed as means ± SD. There was no significant difference between participants visiting family versus in-law group (means) tested with ANOVA.
Fig. 3. Relative change in alpha diversity (Shannon) for participants visiting family versus visiting in-laws.
fecal microbiota alpha-diversity (Shannon-index) compared to participants visiting their own family during Christmas (p < 0.05) (Fig. 3). Furthermore, there was a distinct microbial signature for participants who visited their own family versus participants who visited their in-laws (Fig. 4). This signature comprised seven species with significantly affected relative-change profiles between the two groups (p < 0.05). The microbial signature of participants visiting their own family showed significantly higher variations compared to the in-laws. Furthermore, the biomarker signatures were dominated by the family Ruminococcaceae, showing a significant difference in change in three genera. The genus Rumminococcaceae_UCG-009 exhibited the most divergent outcome: an increase in the family group versus a decrease in the in-laws group. The relative change of two other Ruminococcaceae species (Ruminococcaceae_UCG-002 and Ruminococcaceae_NK4A214_group), was decreased in both groups.
3. Results 3.1. Baseline characteristics We recruited 28 healthy Caucasian participants (14 men, 14 women). Four participants were excluded from the analysis. Two participants did not provide fecal samples on the second time point (T2) due to the “yuck-factor” (a feeling of horror or disgust towards procedures involving poo) [29,30]. One participant did not complete the dietary questionnaires. One participant followed an extraordinary dietary regime and fasted the day before Christmas (December 23rd), with a total intake of only 3 kcal. Of the remaining twenty-four participants that could be included in subsequent analyses, sixteen participants visited their in-laws during Christmas and the remaining eight celebrated Christmas with their own family (Table 1). The relative changes in dietary macronutrients and alcohol consumption during Christmas did not significantly differ between those who visited in-laws versus their own family. Both groups reported a relative increase in saturated fats and proteins, due to a greater consumption of animal based products during Christmas (Table 1).
4. Discussion The multivariate statistical model revealed two distinct biomarker signatures containing seven species that are able to distinguish the participants visiting their family from participants visiting their in-laws during Christmas (Fig. 4). We identified three Ruminococcaceae genera with a different degree of change in both groups. In this regard, the genus Rumminococcaceae_UCG-009 exhibited most divergent outcome with a relative increase in the family group versus a relative decrease in the inlaws group. Strikingly, humans with major depressive disorders [31] and mice exposed to chronic stress [32] are found to have significantly lower levers of Ruminococcaceae genes. These findings could imply that visiting in-laws during Christmas induces high stress levels, which in turn reduces the presence of Ruminococcaceae genus in the gut. We did
3.2. Alpha-diversity and microbial signature Amplicon sequences were processed using dada2 yielding a total of 2354 unique amplicon sequence variants with a minimal sample coverage of 53,340 sequences (Additional file 1). Participants visiting their in-laws had a significant higher change in 3
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Fig. 4. Biomarker signatures for participants visiting family versus visiting in-laws. AUC = 0.78 for the difference between family and in-laws. Value between ‘0’ and ‘1’ reflects a decrease in amplicon sequence variants (ASV), value above ‘1’ reflects an increase.
not have participants report on perceived stress in in-law situations, and it is unlikely that all experienced this feeling of distress. Yet finding a significant decrease in the whole-group analysis could imply an underestimation of the effect size. In the subgroup visiting their own family, we also saw a distinct decrease in two different Ruminococcaceae genes. Potentially not only participants visiting their in-laws, but also participants visiting their family experienced increased psychological stress during Christmas. Furthermore, the model showed higher variations in microbial signature for those participants visiting family during Christmas, versus the group who celebrated Christmas with their in-laws. We know from both animal and human studies that social and physical interactions between individuals promotes both species richness (abundance) and microbial diversity, resulting in a so called pan-microbiome [33,34]. We hypothesise that participants visiting their own family had more physical contact with their relatives compared to the in-law group. which might explain the greater variations in microbial species in the family group. These findings could potentially have clinical relevance, since we know that a higher diversity is one of the most important predictors of host health [35].
supervision from the research-team. Despite the standardized questionnaires, the embarrassing and confronting nature of facing one’s food intake, especially during Christmas, and the inconvenience of filling out a dietary assessment are likely to have influenced the reporting [36]. Differences in the completeness of the forms were noted upon analysis and could therefore have attributed to bias in the results.
4.1. Limitations
Acknowledgments
This study was limited by its observational nature. Participants were free of choice whether to visit family or in-laws during Christmas; this decision was not assigned by the investigators. It is possible that participants with a greater aversion towards their in-laws have used the excuse of participating in a scientific study addressing Christmas poo to not visit their in-laws during Christmas. Since we did not score the level of (experienced) stress during Christmas visits, we can only postulate that the difference in biomarker signature between visiting family and in-laws was due to stress. In addition, multiple (un)known confounders might have influenced our main outcome measures. Beyond known factors like diet, gender, BMI, age, physical exercise, pets and individual microbiome variations, there are many other yet to be identified factors that may have influenced our outcomes. Moreover, this study relied on a small group of volunteers as participants, and analysis of a greater pool of individuals would help to identify trends in microbiota changes with greater accuracy. Finally, in this study participants had to report their food intake, without
We would like to thank all study participants, Han Levels and Maaike Winkelmeijer for fecal DNA extraction, Luuk Scheres, Thijs van Mens, Kristien Bouter, Annefleur Koopen, Jurre Olivier and Robert-Jan de Muinck Keizer for their contribution during the scientific discussions regarding the manuscript.
5. Conclusion This study identified a microbial signature that can discriminate between participants who visited their own family versus their in-laws during Christmas. However, observed differences between the potentially stressful event of visiting in-laws during Christmas and changes in the gut microbiota composition are modest. Before we can recognize inlaws as a new environmental factor that can influence the gut microbiota composition and subsequently host health, a larger randomized controlled study is needed to reproduce these findings and confirm a direct link between visiting in-laws, stress and the gut microbiota composition.
Funding This study was sponsored by the Department of Vascular Medicine of the Academic Medical Center of Amsterdam.
Availability of data and materials The raw sequence data set has been deposited in the European Nucleotide Archive under project PRJEB22340. 4
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Contributors
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NC, MF, HH, AK, JR and MN conceived and designed the project. NC and MN collected samples and performed the experiments. NC, MF, EL, MD, AP, HH, JH and AK analyzed and interpreted the data. NC, MF, EL, MD, JH, HH and MN wrote the paper. All authors commented on the manuscript. All authors read and approved the final manuscript. Ethical approval This study was approved by the Medical Ethical Committee of the Academic Medical Center in Amsterdam, The Netherlands. Consent for publications Consent for publication has been obtained from all participants. Declaration of Competing Interest All authors declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work. References [1] Mirza I, Jenkins R. Risk factors, prevalence, and treatment of anxiety and depressive disorders in Pakistan: systematic review. BMJ 2004;328:794. https://doi.org/10. 1136/bmj.328.7443.794. [2] https://en.wikipedia.org/wiki/Caganer. Extr May 2018. [3] http://www.todayifoundout.com/index.php/2010/12/the-word-mistletoe-literallymeans-dung-twig/. No Title. Extr August 1st 2017. [4] Parker T, Stone M. South Park. The Complete First Season: ‘Mr. Hankey, the Christmas Poo’. 272; 2003. [5] Bouter KE, van Raalte DH, Groen AK, et al. Role of the gut microbiome in the pathogenesis of obesity and obesity-related metabolic dysfunction. Gastroenterology 2017;152:1671–8. https://doi.org/10.1053/j.gastro.2016.12. 048. [6] Gill SR, Pop M, DeBoy RT, et al. Metagenomic analysis of the human distal gut microbiome. Science 2006;312:1355–9. https://doi.org/10.1126/science.1124234. [7] Turnbaugh PJ, Ley RE, Mahowald MA, et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006;444:1027–31. https://doi. org/10.1038/nature05414. [8] Bäckhed F, Ding H, Wang T, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 2004;101:15718–23. https://doi. org/10.1073/pnas.0407076101. [9] D’Aversa F, Tortora A, Ianiro G, et al. Gut microbiota and metabolic syndrome. Intern Emerg Med 2013;8:11–5. https://doi.org/10.1007/s11739-013-0916-z. [10] Arora T, Bäckhed F. The gut microbiota and metabolic disease: current understanding and future perspectives. J Intern Med 2016;280:339–49. https://doi.org/ 10.1111/joim.12508. [11] Devkota S, Wang Y, Musch MW, et al. Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice. Nature 2012;487:104–8. https://doi.org/10.1038/nature11225. [12] Ikeda A, Iso H, Kawachi I, et al. Living arrangement and coronary heart disease: the JPHC study. Heart 2008;95:577–83. https://doi.org/10.1136/hrt.2008.149575. [13] Vuong HE, Yano JM, Fung TC, et al. The Microbiome and Host Behavior. Annu Rev
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