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Funding: European Regional Development Fund (ERDF) “On Implementation of Activity “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”

Project Title: “Investigation of interaction of smoked dietary products with gut microbiome”

Project Nr.:

Period: 36 month (1st January 2018 – 31 st December 2020)

Project costs: 133 806,00 EUR

Project implementer: Dr.biol. I. Kalniņa

Considering the immense choice of harboured enzymes the gut microbiome is a serious contributor to biotransformation of incoming compounds with a prominent role in health and disease. Wood smoke used in processing of food products for flavouring and preservation contains a great variety of compounds with potential to interact with the microbiome. The main objective of the project will be multidimensional investigation of the impact smoked food especially fish consumption exerts on human gut microbiome to gain insight in the responding functional pathways and metabolic consequences. The study will be designed as the crossover dietary intervention including following experimental steps: evaluation of taxonomic and functional composition of the microbiome in humans, detailed characterisation of distinct target communities and investigation of impact of smoke compounds on microbial communities residing in distinct sections of the gut using mouse models. Implementation of the project will give insight in the mutual interaction of bacterial taxa and the role individual members play in the community, as well as add mechanistic understanding on “microbiome to host” metabolic crosstalk. Answering these questions will contribute to the data pool necessary for designing microbiome based tools and consultation on dietary choice to improve healthcare.

Information published 02.01.2018.

Progress of the project

1 January 2018 – 31 March 2018

The project is aimed at investigation of the impact of smoked food consumption on the human gut microbiome. It is expected that the crossover dietary intervention study will provide novel information not only on the response of gut microbiome to the smoked foods, but also wide spectrum of other food items commonly included in daily diet. During the first quarter of the project implementation study specific informed consent forms and questionnaires for diet and lifestyle related information collection have been elaborated. The project protocol has been submitted for consideration to the Central Medical Ethics Committee. Currently planning of the dietary intervention stage and survey of potential participants is in progress. 

Information published 29.03.2018.

Progress of the project

1 April 2018 – 30 June 2018

For evaluation of impact increased smoked food consumption has on the human gut microbiome 26 participants have been recruited to dietary intervention study. Two participants have withdrawn from the study during the second week due to the factors incompatible with requirements of the study. The study is designed to contain three week-long intervention periods concurrently covering all types of target diet (fish, smoked fish and smoked ham) intervened by washout periods two weeks each. Dietary intervention phase of the project will be completed by the end of the august. General phenotypic information from the participants who continue the intervention has been collected using LGDB and project specific questionnaires comprising questions on medication, diet as well as other lifestyle factors. Background lifestyle factors during the intervention are controlled using standardized inquiry forms filled up throughout the study. The study protocol has been approved by Central Medical Ethics Committee of Latvia (Nr. 1/18-06-07).

Information published 29.06.2018.

Progress of the project

1 July 2018 – 30 September 2018

Recently dietary intervention stage of the project has been completed. In total 24 participants aged 19 – 62 years (average age 39,5 years) were involved in 11 weeks long study comprising three week-long intervention periods concurrently covering three types of target diet.  The target diet included fish (salmon provided raw and cooked at home), smoked salmon and smoked pork (in both cases smoked with hardwood smoke). During the study participants monitored their daily diet as well as reported used food supplements and medicaments. Blood samples were collected before and after each intervention period for biochemical analysis (complete blood count, lipids and inflammatory markers) and participants donated stool samples for the gut microbiome analysis. Currently samples are under processing for the gut microbiome profiling including high throughput sequencing analysis.

Information published 28.09.2018.

Progress of the project

1 October 2018 – 31 December 2018

During the concluding phase of the first part of the project we have finished sample processing for the gut microbiome analyses. The study participants were asked to donate blood samples for biochemical measurements and samples for gut microbiome according to the eight visit schedule. From 24 participants included in the dietary intervention study the full set of samples was donated by 21 participants. No microbial DNA was obtained from two samples leaving 180 samples for the gut microbiome profiling with high throughput sequencing approach. Concurrently the follow-up data on habitual diets, physical activity and medication on the daily bases were collected using questionnaires to add information on factors interacting with the gut microbiome. Thus the first stage of the project has been completed.

Information published 28.12.2018.

Progress of the project

1 January 2019 – 31 March 2019

Whole-metagenome shotgun sequencing has been selected as the method of choice for assessment of variation in the composition of the gut microbiome induced by dietary changes. Current approach provides information on both taxonomic content as well as functional characteristics of microbial communities residing in the gut covering not only bacteria but also eukaryotes. During the first quarter of the second project phase optimization of the protocols for the specific needs of the study has been performed. For the metagenomic profiling library preparation of collected microbial DNA samples is in progress. It is expected that metagenomic profiling in combination with longitudinal data on habitual diet will not only give insight on the influence of smoked product consumption on processes occurring in gastrointestinal microbial communities, but also will reveal additional information on functional dietary factors essential for our understanding of the gut ecosystem.

Information published 29.03.2019.

Progress of the project

1 April 2019 – 30 June 2019

During the last quarter of the project implementation characterisation the gut microbiome composition has been completed. The gut microbiome profiling in 180 samples donated by 23 volunteers participating in the dietary intervention study was performed with whole-metagenome sequencing using MGISeq-2000 sequencer. Obtained sequencing coverage was at least 22 million raw reads per sample. Quality filtering of reads with trimmomatic tool removed up to 15% of reads. HUMAnN2 pipeline including MetaPhlAn2 tool and Kraken2 software were used for functional and taxonomic profiling of gut microbiome communities. Statistical analysis was performed with R program packages and Galaxy module LEfSe. First data show that limited consumption of smoked products has no obvious impact on the gut microbiome. However several suggestive associations with habitual dietary variables have been observed and are currently under investigation.

Information published 28.06.2019.

Progress of the project

1 July 2019 – 30 September 2019

Currently investigation of interactions between dietary factors, in particular smoked products, and human gut microbiota is continued with in-depth analysis of diverse metadata collected during the intervention study. This includes targeted analysis of specific taxa selected on the basis of their functional characteristics and/or role in the host physiology (e.g. A. muciniphila). A set of metabolites for further analysis will be adjusted to complement analysis of specific bacterial taxa. At the same time the sequencing step including analysis of additional samples collected from several participants for evaluation of stability of gut bacterial communities has been completed.

Information published 30.09.2019.

Progress of the project

1 July 2019 – 30 September 2019

Considering development of improved pipeline for processing of raw sequencing reads, sequencing data obtained on the gut microbiomes have been reclassified. Intensive search through the recently published scientific literature focused on analysis guidelines for data from studies with “non-standard” complex study design has facilitated process of data analysis. We have also drafted a workplan for the next step in the investigation of the gut microbial communities, which will include network construction for characterisation of interactions building the gut ecosystem and the role individual taxa play in it. Preliminary analysis for assessment of long-term variability of the gut microbiome supports the evidence indicating compositional stability at the higher taxonomic levels. Further work is needed to pinpoint specific changes in microbiome profiles and possible causal factors. It is expected that this information will provide informative support for network analysis.

Information published 30.12.2019.

Progress of the project

1 January 2020 – 31 March 2020

Currently ongoing data analysis is focused on investigation of co-occurrence patterns of bacterial taxa and microbial community dynamics based on cross sectional data as well as time – series data using different network construction algorithms integrated in such analysis pipelines as SparCC and CoNet. Data derived from samples available for nine participants collected three years before dietary intervention study including eight time points provides opportunity to compare short-term and long-term dynamics in individual communities with different profiles, for example characterised by high/low abundance of physiologically significant Akkermansia muciniphila. In collaboration with dietician data collected on habitual diet of participants are revised for use in analysis to complement studies of gut microbial community dynamics. Preliminary data have been presented in LU 78. International Scientific conference.

Information published 31.03.2020.

Progress of the project

1 April 2020 – 30 June 2020

To explore the gut ecosystem we continue with investigation of interaction patterns and dynamics of microbial communities based on network analysis pipelines including CoNet, LSA and TIME. Concurrently we also analyse association patterns occurring between taxa representing residents of gut microbiome and different factors linked with gut microbiome profiles using collected metadata. For example, while inner gut microbiome diversity expressed as effective number of species showed no correlation with lipid measures, relative abundance of several species including Enterococcus faecium, and probiotics Pediococcus acidilactici and Lactobacillus reuteri was significantly correlated with subject specific lipid profiles. It is expected that combined analysis including data on phenotype, diet and metabolites will bring forward novel information on gut microbiome stability and factors affecting its profile.

Information published 30.06.2020.

Progress of the project

1 September 2020 – 30 November 2020

The human gut microbiome is a rich, versatile system with multifaceted effects on the host phenotype. Our results support the role of the gut microbiome in regulation of subject specific lipid profiles. VariancePartition analysis showed that approximately 24% of variation in the microbiome composition can be attributed to differences in lipid levels. Apart from previously described findings, more detailed analysis has revealed additional associations. For example health-promoting bacteria A.muciniphila showed correlation with favourable average lipid profiles (lower total cholesterol and LDL levels) after adjustment of linear regression for factors linked with lipid levels including age, BMI and gender. For more detailed analysis we have recently obtained metabolite profiles characterising samples collected at six time points during eleven week study from 23 participants. Spectra of metabolites were acquired with nuclear magnetic resonance (NMR) based untargeted analysis of faecal water samples.

Results describing correlation between the gut microbiome composition and lipid measurements were included in the poster presented online during 22nd European Congress of Endocrinology (eECE 2020).

Information published 30.11.2020.

Progress of the project

1 December 2020 – 28 February 2021

The final phase of the project was dedicated to wrap-up project activities. Major research activities of the project were dedicated to the data collection within the framework of dietary intervention study, profiling of the gut microbiome and characterisation of interactions between the gut microbiome and host related variables. High quality microbiome data along with biochemical measurements were obtained for 23 participants from 180 visits. Although no significant changes induced by diet in gut microbiome composition were observed, further analysis of host related variables revealed correlation between lipid profiles and variation in the gut microbiome. Several probiotic species were positively correlated with favourable lipid profiles including Akkermansia muciniphila, whereas contradictory correlations were observed Parabacteroides distasonis considered to be beneficial to health. However, other studies show the link with obesity in accordance with association with unhealthy lipid profile observed in our study. Research results have been described in manuscript prepared for publication. Apart from that we have finished experiments in anaerobic cultures which completes the project.

Information published 26.02.2021.