The gut microbiome may contribute to susceptibility to developing alcoholic liver disease

Alcoholic liver disease (ALD) is a major public health issue, yet the underlying mechanisms between ethanol consumption and injury to the liver are poorly understood.  Alcoholics vary in their susceptibility to developing ALD and alcoholic hepatitis (AH) despite consuming similar amounts of alcohol.  Taken together, this evidence suggests that other factors contribute to the onset and progression of ALD other than direct toxicity of alcohol.  Intestinal inflammation and pro-inflammatory bacterial products have also been observed in ALD patients and preclinical mice models, and intestinal dysbiosis has been observed in patients with alcohol dependency.  With this in mind, a team of European researchers devised a strategy to demonstrate microbiome dysbiosis as a casual driver of liver injury. 

The researchers transplanted human gut microbiota into germ-free mice, and the mice were then placed on a high-alcohol diet.  Microbiota were harvested from human alcoholic patients with or without AH (or low severity AH).  Mice transplanted with AH-microbiota had marked increases in symptoms of liver disease as compared to those mice that received microbiota transplants from non-AH alcoholic patients.  These include severe liver inflammation (including increases in T lymphocytes and natural killer cells), more necrosis in the liver, and higher intestinal permeability.  Enterobacteria counts were high in sever-AH patients and faecalibacterium genus was associated with AH-microbiota with low severity.  In an interesting spin, the researchers also transferred microbiota from an alcoholic patient without AH to mice with liver lesions.  Interestingly, mice who had received these microbiota displayed a reduction in serum alanine aminotransferase levels and a decrease in liver regeneration, suggesting that these microbiota could even possibly reverse alcohol-induced liver lesions. 

These findings not only support an association between the gut microbiome and susceptibility to developing alcoholic liver disease, but also provide evidence that these bacteria may drive disease onset.  These were important findings that support microbiota-causal effect rather than dysbiosis as a consequence of liver disease.  This data could perhaps promote development of novel diagnostic techniques that assess the gut microbiome or bacterial metabolites of alcoholic patients.  Methods such as manipulating the microbiome as a therapeutic approach for these patients could also be explored. 

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.

Emulsifiers in food cause many adverse health effects in mice

Emulsifiers are often used in ice cream to keep it stable and to give it it's texture.

People often have spirited and impassioned views on the safety and consequences of adding ‘unnatural’ molecules to food.  An important aspect of this debate that everyone must keep in mind is the impossibility of testing every component of every molecule for its safety and long term impacts.  That being said, it should come as no surprise that new research can often teach us about the unexpected and overlooked safety issues regarding many food additives.  The newest class of compounds to come under scrutiny is emulsifiers, and a new paper published in Nature last week shows that these compounds may negatively impact the body through modulation of the intestinal lining and the microbiome. 

Emulsifiers are compounds that increase the stability of an emulsion.  They are often molecules like surfactants that have two parts, hydrophobic carbon chains and hydrophilic polar head groups.  Soap and egg yolks are common examples of emulsifiers.  There  are, of course, chemically produced emulsifiers as well that are often used in food.  Two examples of these, which were the emulsifying compounds used in the study, are polysorbate-80 (P80) and carboxymethylcellulose (CMC), and they are added to all sorts of foods like ice cream and pudding.  Evidence from this paper suggests though, that at least in mice these emulsifiers are wreaking havoc on the gut and microbiome.

A team of researchers from Israel, Cornell, and Emory did a variety of experiments on mice that were fed either of these emulsifiers in their water (at a concentration of 1%, similar to the levels added to human food.)  They first noticed that these mice had greatly compromised mucous layers on their gut, which allowed for bacteria to actually reach and be in contact with their epithelial gut cells.  In these mice gut permeability (leaky gut), inflammation, and incidences of colitis were all increased.  In addition, the inflammatory response and gut permeability were directly related to the average distance of bacteria to the actual epithelial layer; the closer the bacteria the more inflammation and permeability.

The researchers also measured the microbial populations of the feces in these mice and those that were eating emulsifiers had much less diverse microbiomes, which were enriched in Proteobacter, which are known to be associated with inflamed guts, and reduced in Bacteroidales, which are associated with healthy guts.  Also those eating emulsifiers had in increase in Ruminococcus gnavus which is associated with type 1 diabetes, as we have written about in the past. Interestingly, those mice that were given the emulsifiers tended to eat a lot more food than there control counterparts, and this led to weight gain and obesity amongst the mice drinking emulsifiers.  Moreover, these same mice had higher fasting glucose levels, indicative of impaired glycemic control and metabolic syndrome.  The scientists tested if these effects were seen in mice that were fed the emulsifiers in their food, rather than in their water, and the same outcomes were observed.  In addition, the scientists observed shifts in the production of certain short chained fatty acids and bile acids produced by the microbiome in mice fed emulsifiers (click on the tags below to read about the wide range of health effects that both these compounds have been implicated with).

The researchers then did a series of experiments that showed that it was the actual shifts in the microbiome populations and not just the change in mucous that was primarily responsible for the adverse health effects in the mice given emulsifiers.  For example, germ free mice that were given  emulsifiers did not have compromised mucous, and many of the negative health effects like inflammation were not observed.  On the other hand, performing a microbiome transplant from a mouse given the emulsifiers to a mouse that was not given the emulsifiers did result in these negative health effects, including the bacterial penetration of mucous to the epithelial lining.

This was a fantastic article that may, in time, prove to be immensely important.  Of course all the usual caveats apply, such as studies in mice are not indicative of human responses, and more studies must be performed in order to confirm these findings.  Still though, the introduction of emulsifiers into the mice’s diets resulted in many of the negative health impacts that are associated with the microbiome, something that we really haven’t seen in the literature before now.

New research on the timeline and mechanisms of C. diff infections

Protein structure of C. diff toxin B

Clostridia difficile infections are often the subject of this blog, but we rarely ever discuss how the infection actually occurs.  Scientists know that C. diff spores, which are not very uncommon in nature, can enter the gut from a variety of sources.  Once the spores reach the gut they normally just pass through unnoticed.  However, given the right conditions, these spores can take hold, germinate, and grow.  At some point during infection, the C. diff produces toxins which can compromise gut permeability (i.e. cause ‘leaky gut’) which leads to inflammation and all the nasty effects associated with the disease.  The exact gut conditions that trigger C. diff spore germination are not known, but scientists are convinced that the microbiome is involved because taking antibiotics, which wipe out the normal gut flora, make people susceptible to C. diff infection.  Some research has suggested that certain bugs in the microbiome outcompete C. diff for resources.  Other research shows that secondary bile acids, which are produced when the microbiome breaks down bile acids, inhibit C. diff germination.  Scientists are still working hard to understand the mechanisms of this infection, and just this week research out of the University of Michigan, published in Infection and Immunity, has shed new light on the process. 

The scientists first gave a group of mice antibiotics to make them susceptible to infection, and then fed the mice C. diff spores.  After, they euthanized mice every 6 hours to measure the progression of C. diff infection.  They learned that within 6 hours the spores had already germinated and entered the vegetative state in the feces and large intestine of the mice.  Over time, the C. diff progressed their way up the distal end of the large intestine all the way to the stomach, until the entire gastrointestinal (GI) tract was infected.  After 30 hours, sporulation of C. diff occurred, and interestingly this coincided with the production of C. diff toxins.  These toxins were found throughout the GI tract, however, inflammation only occurred in the large intestine, and not in the small intestine.  After 36 hours the infection had become severe enough that all animals were euthanized.

The scientists also measured the bacterial population and bile acid content of the gut during the infection.  After antibiotic treatment the microbiome was drastically altered and Lactobacillaceae flourished.  Once infection took hold the Lactobacillaceae were supplanted by C. diff in the large intestine, although the Lactobacillaceae still dominated the small intestine population, which, notably, did not become inflamed.  Secondary bile acids, which are produced by the microbiome and linked to C. diff germination, were abundant prior to antibiotics.  After antibiotic treatment, the large intestine had fewer secondary bile acids, and in the most infected regions had no detectable secondary bile acids.

This research is the first to develop a timeline for C. diff infection in mice, and strikingly it occurs very rapidly, with symptoms showing within 2 days.  This study also supports the notion that an altered microbiome is critical to C. diff infection, and that secondary bile acids may in fact play a crucial role in keeping C. diff from vegetating.  Interestingly, this study fits in well with a previous study we wrote about that showed the benefits of secondary bile acids in preventing C. diff infection.

Microbiota biofilms and colorectal cancer

Colorectal cancer is the 4th most deadly cancer in the world, and over 1 million people are diagnosed with it each year.  It has very few genetic indicators and it is rapidly growing in prevalence, thus researchers believe it is very likely associated with environmental causes.  An obvious environmental cause would be the microbiome, and researchers from John’s Hopkins helped establish this link with their recent publication in PNAS.  In their article they show that biofilm formation in the colon is tightly correlated with colorectal cancer.

The researchers studied a cohort of people that had colorectal cancer along with healthy people as controls.  In those people with tumors they studied the microbiome of the tumors themselves along with other, distal parts of their colons.  They discovered that the majority of people with tumors, whether benign or malignant, had thick biofilms growing on and even in the tumor.  What’s more, is that the researchers noticed that biofilms were forming all along the colon, even in the distal parts.  Biofilms were not seen in healthy patients, and in some of the patients with tumors. 

Interestingly, the bacteria in the biofilms between different patients did not necessarily correlate, and so it appeared the presence of biofilms, rather than the composition of the biofilm was critical.  Moreover, the biofilms studied decreased the gut permeability, leading to ‘leaky guts’, which we have covered on this blog before.

A normal colon has a mucous layer to prevent any bacteria from infiltrating the underlying epithelial cells.  It is possible that people with decreased mucosal integrity are at risk for bacteria to invade and form these biofilms which may eventually lead to cancer.  In fact, according to this small study, people with biofilm formation in their colon have a 5-fold increase in their likelihood to get colorectal cancer, much higher than any other known indicator.  More research on a larger scale still needs to be performed.  Still though, biofilm detection could be a useful diagnostic for colorectal cancer, and biofilm management could be a target for drugs or probiotics. 

Our beneficial relationship with our virome

X-ray crystallographic structure of a Norovirus capsid.

We have been championing the virome since the inception of the AMI.  We believe that with time, viruses will prove equally as important as bacteria within the microbiome.  To this end, a paper published last week in Nature shows evidence that a specific virus can promote a healthy gut in mice the same way that bacteria do.  The virus, murine norovirus (MNV), was able to successfully restore function to mice with compromised guts.

The authors started with two groups of mice, a control group and a germ-free group.  The control group had normal guts and immune function as measured by gut morphology, and the amount of T-cells.  The germ free mice had thin, leaky guts, and low levels of T-cells.  The scientists infected these germ free mice with MNV and allowed it to proliferate.  Upon investigation of these mice, their gut integrity and immune function resembled the control group.  A second experiment was performed on mice that had been given a course of antibiotics that wiped out the normal microbiome and resulted in an abnormal immune system and compromised gut.  When these mice were infected with MNV they too saw an improvement in health.  In a final experiment mice were given pathogenic bacteria that damaged the gut, but when infected with the virus the negative effects from the pathogens were diminished.

Viruses have a bad reputation, but that’s because we generally only care about the ones that make us sick.  There are countless viruses that exist in our guts though, many that we do not interact with at all, and many symbiotic ones which have yet to be discovered.  It is time that we appreciate the entirety of our microbiome, not just the bacteria but the eukaryotes, archaea, fungi, and viruses as well.