The vaginal microbiome changes during and after pregnancy

The vaginal microbiome is critically important to a healthy pregnancy, and studies have shown that vaginal dysbiosis during pregnancy can lead to infection and preterm birth.  In order to help understand what the microbiome looks like throughout and just after pregnancy, researchers from England performed longitudinal studies on 42 pregnant women.  They published their results last week in Nature Scientific Reports.

The scientists sequenced the microbiomes of the 42 women throughout their pregnancies, and then for the 6 weeks afterwards for some of the women.  They discovered, in agreement with other literature on the subject, that the vaginal microbiome becomes dominated by Lactobacilli species during pregnancy.  The Lactobacilli are thought to prevent pathogens from colonizing the vagina because they produce lactic acid which decreases the overall pH of the vagina, and they secrete antibacterial toxins.  These Lactobacilli are also important as they are normally the first to colonize the new infants' guts after they pass through the birth canal. 

The researchers also learned that the microbiome shifts away from Lactobacilli and towards a more diverse microbiome in the period immediately following birth.  The new bacteria that colonize are often associated with vaginosis, and these can lead to inflammation and infection of the birth canal in some women.  The scientists suspect this shift occurs because there is a sudden drop in estrogen production upon removal of the placenta.  The increase in circulating estrogen is thought to be important for Lactobacilli colonization, so it makes sense that the rapid decrease in estrogen decreases Lactobacilli abundance.

Finally, this study showed that there were geographic and ethnic variations to the pregnant microbiome.  While each microbiome was associated with a healthy pregnancy, there were important differences, especially on the species level.  For example, Asian and Caucasian women’s pregnant microbiomes were dominated by Lactobacillus gasseri, while this species was absent in black women’s pregnant microbiomes.

This paper helps show the normal progression of the microbiome during and after pregnancy.  With the mounting evidence that the microbiome is often a contributing factor to preterm birth and some post-partum diseases, papers like this one are important to some day discovering the mechanistic basis for our microbiome's association with these issues. 

Freezing fecal samples preserves the microbiota

Editors note: Happy St. Patrick's day to all our readers!  We hope you all enjoy some tasty fermented beverages today, (always in moderation based on yesterday's blog), and instead of corned beef and cabbage, how about corned beef and kimchi!

The microbiome field has exploded over the past few years in large part due to the advent of high-throughput sequencing technologies.  These technologies give scientists the ability to sequence the bacteria in a sample at a fraction of the cost with much greater accuracy than prior methods.  With the growth of this new field, there are more research teams conducting microbiome research with each lab doing things slightly differently. It’s important for scientists to understand the multiple factors that influence the results of experiments, and one of those variables is the storage condition of samples prior to DNA extraction. 

A research team from Ireland published a paper in the Proceedings of the National Academy of Sciences (PNAS) that investigated the impact that storage techniques had on the microbial communities within samples using a MiSeq from Illumina. While it is likely that immediately extracting the DNA from a sample is the most ideal method for research, this is often not feasible due to sampling locations as well as collaborations between investigators at various sites. 

In this study, samples were collected from 7 individuals and each sample was separated into three groups, fresh samples that were processed within 4 hours of sampling, samples that were “snap frozen” and immersed in dry ice for 4 minutes before being stored for a week at -80°C, or samples that were frozen immediately at -80°C.  The researchers found that there were no significant differences between the three experimental groups. The samples that were sampled fresh, snap frozen using dry ice, and those frozen only at -80°C had similar numbers of total bacteria as well as bifidobacteria which was sampled due to its sensitivity to freezing as well as its low abundance in fecal microbiomes. 

This study has shown that immediately freezing fecal samples should appropriately preserve them for use in research. This type of study is incredibly valuable in order for the greater scientific community to understand the impact that important variables such as storage techniques can have on microbial sampling.  There are many variables that play a role in microbiome data and it is important for studies like this as well as initiatives like the Microbiome Quality Control Project to lead the way in allowing us to better understand these factors.  

The microbiome of alcoholics may contribute to pathologies

We have written before about the microbiome’s association with alcoholism, and how it has been implicated in many of the maladies connected with the disease.  Recently, research out of George Mason University, published in PLoS ONE, explored the molecular mechanisms behind this relationship.   The scientists measured the metabolites that were formed by the microbiome of alcoholics and compared it to healthy controls.  They discovered that the metabolites that differed between the two groups have important implications on gut health.

The scientists measured the volatile molecules that were being effused from the feces of 18 healthy controls and 16 alcoholics.  The alcoholics’ feces contained high levels of an organic compound called tetradecane, which is known to cause oxidative stresses.  Increased oxidative stress in the gut, especially in alcoholics, is associated with increased gut permeability (i.e. leaky gut), and alcoholic steatohepatitis (i.e. a type of liver disease).  Moreover, specific fatty acids, which are known to reduce oxidative stress (antioxidants), were more depleted in alcoholics when compared with healthy controls.  In addition, the alcoholic feces consisted of lower abundances of short chained fatty acids (SCFAs), which are nearly always associated with intestinal health (click the SCFA tag below to learn more).  Finally, other molecules which are associated with health, like caryophyllene and camphene, were decreased in the guts of alcoholics.

Overall these results show the possible mechanisms by which the microbiome contributes to alcoholism.  Specifically, it appears that the alcoholic microbiome may create oxidative stress molecules, which contribute to gut toxicity.  In addition, the scientists suggest this work could be used as an alcoholism diagnostic, as the characteristic metabolites between the groups were statistically significant.

New study may lead to microbiome-based colon cancer diagnostic

Colorectal carcinoma, or colon cancer, is one of the most common cancers and a leading cause of death among the elderly. The cancer is often caused when adenomas, benign tumors, transform into malignant tumors called adenocarcinomas. The gut microbiome has been long implicated in colorectal cancer however it is not yet clear how the microbiome integrates with other risk factors that may lead to this cancer in patients. A study published in Nature Communications studied various risk factors that may influence this progression into colon cancer. 

The scientists collected stool samples from 156 individuals including healthy individuals as well as those with colorectal adenomas or carcinomas and sequenced the microbial DNA. They identified specific bacteria that were different in patients with adenomas or carcinomas and healthy individuals. For example, Bifidobacterium was deficient in those with benign or malignant tumors as well as other bacteria that are signatures of a healthy microbiome.  This showed that the microbiome was different between patients with the cancer and those without.

The scientists also studied whether they could use the microbiome as a diagnostic tool for identifying patients with carcinomas. Using the data they collected of bacteria in the guts of patients with  and without tumors, they came up with a set of markers that could detect the presence of carcinomas. They also specifically investigated whether this could be done for adenomas, which are harder to screen for than carcinomas but important to identify at an early stage in order to intervene before they mutate into carcinomas. This was also successful, though further investigations would most likely be needed to identify adenomas. 

In addition, the researchers looked at what impact diet had on the microbiome of patients with and without carcinomas and adenomas.  They found that those individuals eating higher levels of red meat had carcinoma enriched bacterial communities in their gut and those eating fruits and vegetables had lower levels of carcinoma enriched bacteria.

Continued research into the role that specific risk factors like smoking, diet, and obesity have on the microbiome will help us better understand how these factors influence the onset of this cancer. In the future, it may be possible to utilize the microbiome as a diagnostic tool for the identification of either colorectal adenomas or carcinomas, or even to help develop new microbiome based therapies for colorectal cancer. 

The microbiome and its influence on evolution

The interaction between a microbiome and its host species, and the impact of this interaction on both bacterium and host organism evolution, is a fascinating subject.  Known as the hologenome theory of evolution, it has been widely understood that microbiome communities and their animal hosts undergo natural selection in concert.  In other words, human and microbiome evolutionary processes are not mutually exclusive.  In the hope of gaining a deeper understanding of this relationship, a recent study published in Nature Communications examined the exact role of the microbiome in host speciation of a vertebrate organism.  Researchers explored evolutionary divergence within two subspecies of mice and examined to what extent genetic loci played a role in regulating intestinal microbiota populations. 

Four strains of mice were used to develop a unique mouse model paradigm.  Researchers collected two naturally occurring and genetically distinct mouse populations – M.m. musculus and M.m. domesticus –that are geographically isolated in Europe (see map).  Interestingly, a small “hybrid zone” exists in which both subspecies interbreed.  Researchers examined these three distinct populations and added a fourth by artificially producing a hybrid strain between both subspecies under strict laboratory conditions.  The purpose of the laboratory-bred hybrid was to control for any confounding influence from environmental factors (it has been demonstrated that environment can influence microbiome populations and gene expression).  A subsequent analysis was performed to investigate intestinal microbiome genetic profiles for both pure species and hybrid species in addition to gene expression with potential connection to pathological indications in host species. 

Pyrosequencing techniques of a bacterial rRNA gene were used to identify genetic differences between pure species and “naturally occurring” wild-hybrid species.  Bacteria community structure varied significantly in hybrid generations, both those obtained from the hybrid zone and bred in the lab.  The most significant differences were observed in the laboratory-bred hybrid generation.  Furthermore, there was a significant decrease in bacterium species richness in both wild and lab hybrid mice.  QTL mapping, a technique used to assess underlying genetic factors, displayed a low number of microbiota genomic loci present in both hybrid strains.  This finding was thought to explain a 14.1% variation in bacterial community structure, ultimately suggesting that genetic deficiencies disrupted microbiome communities. 

QTL profiling of microbiota also indicated a high frequency of genes implicated in immune system regulation.  Laboratory hybrid generations were shown to have imbalanced T-cell subset populations at various immune sites (e.g. spleen, mesenteric lymph nodes) in combination with the aforementioned alterations in bacterium community structure.  Additionally, histological analysis of intestinal mucosa revealed significant increases in inflammatory cell infiltrates and epithelial ulcerations in both wild and lab hybrid mice as compared to both purebred wild species.  The investigators suggest a discrepancy in communication between microbiome populations and the host immune cells. 

The data from this study suggest abnormal genetic architecture and irregular immune function result in an altered intestinal microbiome due to cross-breeding between two vertebrate subspecies.  The hybrid species displayed poor fitness for survival.  As suggested by the authors, species divergence, and the consequentially genetic disruptions resulting in an altered microbiota population, is perhaps responsible for generating subspecies population isolation.  This theory offers a possible explanation or contributing factor as to why M.m. musculus and M.m. domesticus populations are geographically distinct.  Obviously, evolutionary biology is an enormously complex process with many diverse and distinct influences.  However, as this article proposes, perhaps the microbiome has had a much more profound influence on evolutionary fate than we previously realized.

Queen bee microbiome distinct from worker bees

Bees are essential to agriculture because of their role in pollination. Recent concerns about declining bee populations, especially that of the European honey bee, Apis mellifera, has increased researchers’ interest in the microbiome of the honey bee. The microbial composition of worker bees, as well as the queen-rearing process, is well known, but little to nothing is known about the queen bee microbiome. Researchers from North Caroline State University, Wellesley College, and Indiana University analyzed the queen bee microbiome composition at different stages of development, and compared their findings to the worker bee microbiome composition.  Their results were recently published in Applied and Environmental Microbiology.

 At North Caroline State University’s Lake Wheeler Honey Bee Research Facility a single honey bee colony was chosen as the source of all queen bees used in this experiment, with the purpose of eliminating genetic variance as a variable. The queen bees had their gut microbiomes tested at five different stages of development: larvae, newly emerged queens, maturing queens in the mating nucleus, and laying queens both before and after their offspring emerged.

 Drastic changes in the microbiome composition occurred during the maturation of queen bees, with the largest changes occurring between young queens (larvae and the newly emerged adults) and mature queens.  The scientists speculated that these changes occurred because during maturation the queens begin to physically contact worker bees. To test this, the researchers characterized the microbiome of worker bees that may have helped rear the queens. The worker bees exhibited a similar communal microbiome, while the queen bees appeared the lack this microbiome; instead they showed a predominance of α-proteobacteria. In fact, there was greater variability among related queen bees than there was among unrelated worker bees. The scientists think this may be because of the communal environment that worker bees are exposed to, from which queen bees are carefully secluded.

 The main question here is where do the queen bees get their microbial characteristics from? Human’s and other mammals’ microbiomes are greatly influenced by the mother during the birthing process. Conversely, queen bees appear to develop their microbiome because of the “royal jelly” they are carefully fed during development, which comes from the hypopharyngeal glands of nurse worker bees, and because of the care worker bees take in keeping the queen’s environment clean by grooming and disposing of her fecal matter.

 The most significant finding from this study is that the queen bee’s microbiome does not reflect the specific microbial community of her worker bees. This may not seem important to a non-beekeeper, but to someone who works with bees this means that the movement of queen bees from one colony to another should not disrupt the microbiome, and therefore the health of the workers. This is good news to beekeepers and those in the agriculture business who are concerned with the health of their bee populations, especially in light of the recently declining bee population.