Microbiome metabolite, TMAO, associated with increased risk of chronic kidney disease and atherosclerosis

Our microbiome is responsible for producing many of the molecules and metabolites that end up in our bloodstream.  Here, these molecules can serve many important therapeutic functions, like in the case of short chained fatty acids (SCFAs).  However, some of these metabolites are beginning to be linked to various diseases.  The best known example of this is microbiome mediated production of trimethylamine-(N)-oxide (TMAO).  TMAO is produced by the microbiome from molecules called carnitine and choline, which come from foods like red meat and eggs.  A lot of literature is now being published that is establishing a connection between TMAO and atherosclerosis, and more recently between TMAO and chronic kidney disease (CKD).  In a new study published last week in the Journal of the American Society of Nephrology, researchers from the University of Kansas assessed the connection between each of these things, TMAO, atherosclerosis, and CKD.

The researchers hypothesized that patients with chronic kidney disease would show increased levels of atherosclerosis because circulating TMAO cannot be cleared from the blood by their injured kidneys.  They measured the degree of CKD and plasma TMAO levels in over 100 patients and confirmed their hypothesis: they noticed a direct relationship between severity of their CKD and circulating TMAO levels.  In addition, the researchers noticed that those people who received kidney transplants had improved TMAO levels, showing that the relationship between CKD and TMAO was most likely due to loss of kidney function.  Finally, the researchers measured biomarkers for atherosclerosis in these patients and correlated the amount of TMAO with the amount of atherosclerosis, even showing that the long term survival of these patients was related to the amount of circulating TMAO levels.

This paper is one of the first to make a connection between circulating TMAO levels and kidney function, which itself is related to atherosclerosis and mortality.  The microbiome is ultimately responsible for the creation of TMAO in the blood, by breaking down things like meats, eggs, and nuts.  It appears that a dietary shift, or a modulation of the microbiome that decreases its ability to perform this transformation should be considered for patients with CKD who are at risk for atherosclerosis.

The gut microbiome shift during pregnancy is related to the mother’s secretor status

Fucose chemical structure

An estimated 20% of women of European descent are not able to produce mucous that have fucose sugars attached to the ends of their mucin molecules.  These women are called ‘non-secretors’, as opposed to ‘secretors’ who can fucosylate their mucins.  This rather peculiar genetic anomaly is not appreciated until it is looked at under the lens of the microbiome.  Many of the microbiota in the gut feed off the host’s mucins for energy, and the lack of fucose is a major factor in dictating which communities can survive in their guts.  During pregnancy the mother’s gut microbiota undergoes a dramatic shift, although what variables are important in determining this shift remain unknown.  Last week though, researchers from Finland showed that secretor status was an important indicator in how a women’s gut microbiome shifts during pregnancy.  They published their results in PLoS ONE.

The researchers sampled the gut microbiome of 71 women throughout their pregnancy, and compared it to the secretor status, as determined by genetic testing.  In the first trimester of pregnancy each women, secretors and non-secretors alike, had similar diversities in their gut microbiota.  However, by the third trimester the non-secretor’s gut microbiomes were much lower than their secretor counterparts.  When the scientists measured specific phyla, they observed an increase in the abundance of Actinobacteria in the secreting women, and an increase In the abundance of Proteobacter in the non-secretors.

The changes in gut microbiota in these women may be very important to the microbiome of the infant that is born to them.  As an infant passes through the birth canal he or she is exposed to the mothers’ vaginal and gut microbiota, and these bacteria serve as the initial populations that seed the infants’ own guts.  In addition, some of these specific bacterial populations, such as Proteobacter, are implicated in diseases like IBD.  If these bacteria persist in the mother after birth they may explain the onset or increased risk of some of these diseases.

Antimicrobial peptide can reduce acne infection in mice

Chances are you have experienced some form of acne, or know someone who has.   Also known as Propionibacterium acnes (P. acnes), these gram-positive bacteria drive the pathogenesis of what we commonly refer to as acne.  The bacteria aggregate in high numbers on the skin in areas such as hair follicles.  When bacteria density gets too high, the innate immune system reacts, resulting in a battery of proinflammatory signals and/or reactions.  Current treatments include both topical and oral antibiotics, but these therapies collectively have shown minimal efficacy at the expense of some side effects.  A recent study conducted by researchers from South Korea and Denver sought to explore the use of a novel and custom made antimicrobial peptide (AMPs) called P5.  AMPs have been previously shown to effectively kill bacterial pathogens while at the same time modulating host immunity.  The aim was to see if this tiny cationic peptide sequence could protect against P. acnes induced immune responses both in vitro and in vivo, and therefore present itself as a potential novel acne treatment. 

The researchers first demonstrated that P5 had antibacterial effects in vitro (administering the peptide to bacteria culture dishes).  In a follow-up in vitro experiment, P5 was shown to disrupt the morphology P. acnes, as displayed by scanning electron microscopy images.  P5 was also able to inhibit proinflammatory signals brought on by P. acnes, including reductions in cytokine signaling and toll-like receptor expression in human skin cells.  Furthermore, P5 reduced the expression of a gene responsible for regulating transcription of these inflammatory signals. 

In the in vivo experiments, mice were inoculated with P. acnes and split into two groups, either receiving the P5 AMP or a vehicle control intradermally (in the skin).  P. acnes growth was visibly reduced in the ears of mice when administered P5.  To demonstrate the effective quantitatively, the researchers measured ear thickness to record differences in swelling.  P5 was able to significantly reduced ear thickness in P. acnes mice that had been given P5.  Importantly, P5 alone– i.e., in mice without the P. acnes infection – did not have any effect on inflammation, suggesting the peptide’s role in modulating immunity specifically with P. acnes. 

Treating bacterial infections at the biological level using unique features of bacteria offers a promising technique to combat common maladies such as acne.  In fact, companies such as AOBiome are already utilizing unique methodologies, as their products are designed to help the skin microbiome.  In light of our previous discussions regarding misuse/overuse of antibiotics, this method could offer a much safer alternative toward treating acne.

Misconceptions about antibiotic use remain prevalent

While today’s post is not specifically about the microbiome, it is about a critical issue that we talk about all the time on this blog, the misuse of antibiotics in today’s society. A group of researchers compared the attitudes of parents with children on Mediciad managed care plans to those on commercial plans about the use of antibiotics and published their results in Pediatrics. They utilized insurance type as a proxy for sociodemographic factors as Medicaid is a government run insurance program for lower-income families.

The researchers surveyed approximately 1,500 families in Massachusetts with a child younger than 6 and asked them about the use of antibiotics for their children. Those insured by Medicaid answered more questions about antibiotics incorrectly than those with commercial plans. Another important finding was that those insured by Medicaid were more likely to request antibiotics be prescribed to their child unnecessarily.

The overuse of antibiotics in children can lead to significant health issues during childhood as well as later in life. Many studies, including many by Dr. Marty Blaser, have shown that antibiotic use has critical impacts on the microbiome with many downstream heath effects. Overusing antibiotics can also lead to antibiotic resistance, a rapidly growing public health concern.

A key takeaway from this study is that education by providers to parents about what antibiotics are useful and not useful for is critical. Pressure from parents of sick children will often lead to the unnecessary and ineffective prescription of antibiotics and it is important that these practices are ended. The authors of the paper state,“Tailored efforts for socioeconomically disadvantaged populations remain warranted to decrease parental drivers of unnecessary antibiotic prescribing.”

Different diets can affect C. diff infection and survival

We’ve covered the topic of Clostridium difficile infection extensively on this blog. We know that infection of this bacteria (CDI) can be nasty, sometimes even leading to death. A lot of research has been done to find ways to treat the effects of C. diff infection or to find out how the infection is acquired, but few papers have investigated dietary interventions to help treat CDI.  A new study published by PLoS One examined this, studying how different diets, and specifically how protein content of those diets, affected the severity of CDI.

          7-8 week old male mice were weighed and separated into five groups, each given a different diet. One group was fed a protein-deficient 2% protein diet, and a counterpart group was fed a 20% protein diet. Another group was given a Research Diets regional basic malnutrition diet, while its counterpart was fed a matched control. A fifth group was fed a traditional (corn, wheat, soybean) diet, and acted as the positive control. Mice were fed the diets 12-14 days before being given antibiotics and then infected by C. difficile.  After infection, the mice were housed individually to prevent being affected by other mice. Stool and colon samples were collected from the mice up to two weeks after infection, and the bacterial content was sequenced.

Mice on the 20% protein diet showed delayed onset disease with a 25% survival rate over 2 weeks. Mice on the 2% diet also showed delayed disease onset and had a survival rate of 57.1% over two weeks. A significant statistically difference in survival and weigh loss was seen between the traditional diet and both the 20% and 2% diets, however the survival difference between the 20% and 2% groups was not significant.

In another part of the study done by the researchers, they chose to examine the presence of four gut microbiota groups ( Firmicutes, Bacteroides, Enterobacteriaceae, and total bacteria), after antibiotics or no antibiotics. All aspect of the experiment were the same except for this one factor. In mice not given antibiotics, the total number of bacteria was greater in the colon of mice given traditional diet. In mice given antibiotics, traditional diet-fed mice had lower levels of Bacteroidetes but higher levels of Firmicutes and Enterobacteriaceae. The most significant finding the researchers may have made however, is that traditional diet-fed and antibiotic given mice had the highest levels of C. difficile and therefore C. difficile toxins.

In the end, it can be assumed that diet does influence rates of C. diff infection. The presence of antibiotics also alters the bacterial compositions of the colon, with lower protein diets seeming to protect against or prevent full potential C. difficile infection.

Stool consistency should be considered during microbiome research

Stool sampling remains the most common method of measuring the microbiome of the GI tract.  Researchers are well aware of its limitations, but its ease and convenience for both scientists and donors makes it nearly irreplaceable at the moment.  The most common issue regarding stool samples that is often pointed out is that it is not representative of the GI tract, and that it only samples the lower colon and not the more proximal GI tract.  In addition, it does not account for bugs that are attached to the mucous linings of the intestine rather than those that transiently pass with our feces.  Related to this point, last week Jack Gilbert and John Alverdy, professors from the University of Chicago, published a piece in the journal Gut regarding stool microbiome sampling and stool consistency.

Professors Gilbert and Alverdy argue that stool consistency greatly affects the stool microbiome populations.  The stool consistency is normally a function of intestinal transit time, with the shorter the duration between eating and passing stool being associated with watery stool, while a longer duration is associated with a more solid stool.  They point to studies that that show different bacteria have evolved to either grow rapidly when the stool is quickly moving through the lumen, in order to proliferate with the shorter duration access to nutrients, or to grow slowly and more completely utilize the available nutrients when the stool is accessible for longer periods.  Measurements of stool consistency are hardly ever performed during normal sampling, and these same studies tend to make generalizations about different phyla, like Bacteroides and Ruminococcacea, when in fact these different can be explained by stool consistency. 

In a time where microbiome diagnostics are starting to be considered as helpful indications for varous diseases, this type of quality control needs to be established.  Stool sampling is not perfect, but it is necessary, and for that reason steps should be taken now to improve and control its usefulness, especially in a clinical setting.