How does diet affect our gut microbe health? - Microbiome composition of Boozy Flies by measured by next-gen deep sequencing.

With further awareness of the intricacies of our bodies as the product of not only human but also microbial cell populations, the interplay of microbial contributions to health has been pushed to the forefront of popular science. While our microbiome is tailored to each individual and may help maintain health, many factors can alter microbial composition which may lead to disbiosis: the unbalance of microbial populations that may lead to downstream disease or pathology. With many University student’s diet being supplemented with regular drinking, we are interested in the effect of an ethanol supplemented diet on gut microbiome composition. To elucidate this question, will use a fruit fly (Drosophila melanogaster) model fed with either conventional or alcohol supplemented (0.5% Ethanol) diets for 1 week. Microbial populations will be determined using deep sequencing of bacterial 16S DNA and compared between the two diets.

Previous studies in mice have found significant shifts in abundance of different microbial populations such as Bacteriodetes and Firmicutes in addition to Proteobacteria, Actinobacteria and Corynebacterium in response to alcohol supplemented diets, although these animals were fed a high alcohol diet (5% ethanol) to simulate chronic alcohol pathology. This equates roughly to a bender of standard 5% ABV beer for 7 weeks, and while sounds fun, hopefully is not representative of the standard drinking experience. We expect to see shifts in similar microbial populations in our boozy flies, though likely to a much lesser extent than seen in previous pathology based models.

Methods

Flies and modified diets
Vials were seeded with 10 Drosophila melanogaster flies. Only virgin female flies were chosen to prevent gender biases as well as population expansion. Vials were raised on either a chemically defined agar based holidic diet, or a holidic diet containing 0.5% ethanol at 25˚C for 14 days. Flies were then sedated and euthanized at -20˚C.

Isolation amplification and sequencing of bacterial 16S DNA
Homogenization and DNA purification using an Ultraclean Microbial DNA Isolation Kit following slightly modified manufacturer’s protocol. Bacterial 16S DNA was then amplified via conventional PCR using primers specific for a broad range of bacterial, but not fly 16S DNA. PCR products were quantified using Quant-iT high-sensitivity DNA assay which measures dsDNA concentration based on fluorescence of intercalating dye. Amplified DNA was tagmented (fragmented and tagged) with 2 sets of indexes specific for each group’s library. Fragments were sequenced using an Illumina sequence by synthesis multiplexed cluster technique. Unfortunately, a mechanical failure occurred during the sequencing run, so data analyzed was provided by Dr. Edan Foley from a previous experimental trial run on male flies. Sequence data was given to Dr. Bart Hazes, and analyzed for microbial diversity using Unix based bioinformatics programs and databases. Illumina sequence by synthesis is explained in detail in the video below from Illumina.
                                          

Results and Discussion
It is commonly thought that the composition of the gut microbiome is incredibly important for host health. With the huge amount of high sugar or fat content foods readily available alongside the myriad diets coming in and out of fashion, it is important to determine the effect that nutrition has on our gut flora. Specifically, the effect of alcohol on microbial populations interested us greatly, being among one of the most common nutritional additives to diet relative to the student lifestyle.

Upon sequencing gut microbes from flies fed an alcohol supplemented diet, we found that overall, the diversity of gut microbiome decreased (Figure 1). This drop in diversity was calculated quantitatively using Shannon diversity scores which take into account not only number of species, but relative abundance of each (Table 1). Control flies fed a ‘holidic’ diet with a known composition yielded a score of 5.92, while flies fed a holidic diet with 0.5% ethanol decreased the Shannon score from 4.613.
 

Figure 1. Gut floral composition of ethanol diet Drosophila by 16S DNA sequence. Male Drosophila melanogaster flies fed either holidic control or alcohol supplemented diets for 7 days and then euthanized. Bacterial 16S ribosomal DNA was isolated from whole fly homogenates are amplified using conventional PCR. Bacterial DNA was then tagmented with known adapters prior to Ilumina sequence by synthesis. Identity and proportions of bacteria were calculated based on abundance and sequence identity. a) Proportion of bacterial population on family levels. b) Proportion of population on genus level. Teal indicates population consisting of 39 various Acetobacterea genus'. Other colors correspond to numerous other small microbial populations.

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Table 1. Diversity indexes of gut flora in flies fed an alcoholic diet. Shannon diversity scores were calculated using operational taxonomic units generated by analysis of deep sequencing data. Higher scores are achieved by a combination of variety and abundance of individual species. A score of 0 reflects a purely homogeneous population. Experimental data derived from given data, paper data derived from an upcoming publication from the Foley lab.

When looking at the effects of alcohol on specific populations of gut bacteria, we found a ~20% increase in population of members within the Acetobacteraceae family. This population consisted largely of Acetobacter species (52% and 63% of control and alcohol groups respectively) with the remaining populations consisting of 39 various other genus’. These results are not surprising, as acetic acid bacteria are able to utilize oxidation of ethanol into acetic acid. In addition, the vast majority of other bacterial populations decreased in size. This result was again unsurprising, as ethanol is toxic to most cells that are unable to metabolize it. While low concentrations of 0.5% may seem insignificant, long term exposure may have had the combined effect of increasing the growth of ethanol oxidizing bacteria (ie. Acetobacter) while slowing growth of other species. 

Although it’s hard to point out the biological significance of these results with looking solely at microbial composition, there are several important implications that could be made. The decrease in microbial diversity may have long term effects on nutrient access/ absorption, as many vitamins and other nutrients required for optimal growth are microbial metabolites. Reduction or depletion of these populations may also deplete the host of their nutritional benefits.

Significantly, Shin et all found that Acetobacter species modify Drosophila insulin and insulin growth factor signalling which regulate a huge range of physiological parameters such as body size, metabolism and rate of development (2). Thus, the effect of alcohol on microbial populations likely plays a role in the development of embryos and young children that require precisely coordinated signals while growing. It seems possible that the effect of fetal alcohol syndrome may be, at least in part, due to altered host-microbiome cross-talk. Additionally, depletion of microbial diversity may be affecting myriad possible interactions between resident gut microbes and the host that have yet to be described which could have varying impacts on an equally vast array of physiological parameters.

This experiment highlights the drastic changes in gut microbe composition that can be caused by even small changes in diet. Although this experiment provides important information of the impact of alcohol on the microbial populations, it does not provide any insight into what impact these population shifts have on the host. It is also important to realize that individuals within heterogeneous populations can have a very different gut microflora, and that each microbiome may act differently to identical changes in diet, especially given the complexity of interactions between the host and it’s commensals.


Technical discussion

Although the calculated Shannon scores for our experiment are 5-20 fold higher than those seen in the Foley paper, the trends seen are the same, strengthening the robustness of our results. The difference in magnitude is likely due to the way the data was analyzed. Each bacterial 16S DNA sequence is included into an Operational taxonomic unit (OUT), since sequence doesn’t necessarily describe a species. Stringency of OTUs can be adjusted to be more precise, giving more clusters of association, or less stringent, giving clusters of more broadly related microbes. We obtained 954 individual OTUs across all samples using a stringency of 97% sequence resemblance, which is ~30 fold higher than the 30 species known to be Drosophila gut commensals. This huge number is likely what increased the magnitude of our diversity scores and was likely caused by using whole fly homogenates where all external bacteria, commensal or otherwise, would be included. Homogenized isolated fly guts used in the Foley paper would include only gut microbes and likely a much smaller number of OTUs, though total OTUs obtained in their results was not included in the paper. Another reason is possible contamination from processing samples outside of a biosafety cabinet.  In addition, since only bacterial 16S ribosomal DNA was amplified, the effect of alcohol on eukaryotic commensals such as yeasts remain unknown.



Now with the final post of my MMI 590 blog, its time to embrace the student lifestyle and transition this experiment to ‘human trials’ with a pint or two.

Cheers!

Mike Wong
University of Alberta


1. Bull-Otterson et al. Metagenomic Analyses of Alcohol Induced Pathogenic Alterations in the Intestinal Microbiome and the Effect of Lactobacillus rhamnosus GG Treatment (2013). PLoS ONE. DOI: 10.1371/journal.pone.0053028

2. Shin et al. Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling. Science. Vol.334 no. 6056 pp. 670-674 DOI: 10.1126/science.1212782