The human microbiome or the genome of microbiota closely associated with humans has been under the limelight for quite some time. It has long been known that the human body harbors many different microbes - inside and on the outside. The human skin, hair, mucosal linings, fluids, orifices all provide great environments for a number of bacteria to flourish. The most abundant of these are probably even the most studied are the microbes that dwell in the human gut. To just give you a perspective of the scale of this colonization - let us consider some numbers. It is estimated that the human body harbors thousands (500-1000 species in the gut alone) of bacterial species and that we may in fact be carrying more bacterial cells in us than our own. It is roughly estimated that there are atleast 10^14 bacterial cells while there may be only about 10^12 - 10^13 of our cells in that body that we strive to protect and maintain.
To me this is a fascinating thought.
Now the role of these microbes has long been speculated and never really understood. The primary reason being it is technically very challenging to breed animals in such extreme isolation and then to determine the composition of our microbiome. Technically, we couldn't analyse bacteria that we couldn't grow in culture and despite all this research there are only a handful of bacterial species that we can grow in our labs. But then there came the sequencing revolution. And as genomes got sequenced in days and now hours with low costs and high efficiency, we could now finally identify the bacteria that colonize us. Today in the era of "omes", microbiome is yet another genome scale enterprise and unlike some of the others, it is giving us some startling revelations.
The composition of our gut microbiome has been seen to play a role in a large variety of physiological phenomena from obesity, to diabetes, to nutrient absorption, to infection and immunology. While certain bacteria cause infections and chronic inflammation leading to diseases like cancer, most are commensals or actually symbionts as we now are realizing. Over the past few months, the scientific literature has been bombarded with studies that delineate the role of these gut microbes and their association with our health and disease. The clinical success of fecal transplants and their ease and efficiency, also strengthen the future potential of these studies. Because if some of the evidence is to be believed, a lot of our diseases can be remedied by simply altering the microbes in our gut.
This post deals with one of the recent papers in this arena which shows that sub therapeutic antibiotics can alter the murine colonic microbiome and adiposity of laboratory mice.
Published in the recent issue of Nature, this study does raise some interesting questions.
This study deals with one of the oldest controversies arising from antibiotic use in animal husbandry. For almost half a century now, it has been widely seen and reported that sub-lethal (for the bacteria, of course) doses of commonly used antibiotics promoted the growth of farm animals. Vertebrates across the board - from mammals (goat, sheep, cows) to poultry (Chicken, turkey) exhibited upto 15% of growth and increased weight gain when administered with sub-therapeutic doses of antibacterial agents (penicillins, macrolides, tetracyclines, ionophores etc). Since this effect was not agent specific, and was not shown by anti-fungals or anti-virals, the underlying mechanism was not clear. Since, the doses were sub-lethal, it did not kill the gut flora and therefore do the obvious.
On the other hand, this use of sub lethal doses had stirred up a lot of public anxiety and fear about the rise of resistant bacteria and their spread to humans. While there was not direct evidence for this, the fear was justified to some extent. Mechanisticaly, it has been speculated that these sub-therapeutic doses might suppress the growth of toxin producing microbes and the sub-clinical infections particularly in the post-weaning period, which might indirectly reduce the energy invested on gut tissue replacement and immune response. But this was again never really experimentally tested.
In the recent issue of Nature, Martin Blaser's group at the New York University school of medicine do just that in trying to answer this decades old question.
They exposed young mice at weaning to sub-therapeutic doses of antibiotics like penicillin, vancomycin, penicillin plus vancomycin, chlorotetracycline (STAT mice) or no antibiotic (control) in their drinking water. Seven weeks later then found that while there was no overall difference in the weight or size of these two groups of mice, there was however a over difference in the fat content of the two groups. The STAT mice had significantly higher levels of fat accumulated in them though they weighed the same and consumed the same amount of feed. The STAT treated mice also showed no significant differences in their feed efficiency - weight gained per unit of food consumed. Careful studies showed that while the animals treated with STAT showed an early growth spurt, the actual changes in fat mass began to diverge between the two groups only by week 16-20. In order to understand the events leading upto this they then analyze the animals for the levels of the various hormones that regulate appetite or satiety like leptin, ghrelin, peptide YY etc and they find no significant differences.
They then examined the microbiome of the two groups to determine if the sub-therapeutic antibiotic treatment is leading to these metabolic changes by affecting the GI tract microbiome. And interestingly, they find that while the STAT did not lead to substantial changes in the overall microbial census, it did however lead to alterations in the composition of the microbial population. They find that in the fecal and caecal samples, there is an increase in the proportion of firmicutes vs the bacteroidetes upon STAT treatment and they propose that this taxonomic alteration is making the gut more efficient at extracting energy from the diet. Now, to my mind, while there is a significant change in the composition of the gut microbial community (bacteroidetes/firmicutes), the extent of this change upon different antibiotic treatments does not appear to correlate very well with effect of each antibiotic on the weight gain itself.
(As can be seen in these graphs, while Penicillin causes maximum increase in the body fat % it only causes modest (but significant) changes in the alterations of the gut microbiota (Bateroidetes/ firmicutes). Although I do realize that it is difficult to find perfect correlations in most biological phenomena, I would have liked to see better correlation between the two graphs.)
This is based on previous work where ob/ob mice which are genetically prone to obesity were found to harbor an increased population of firmicutes. They further find that STAT treatment affected the expression of genes related to short chain fatty acid (SCFA) synthesis which play a central role in colonic metabolism. And while different antibiotics affected SCFA genes in different ways, the overall effect was conserved. They validate their hypothesis further by showing increased levels of these fatty acids in the caecal contents of STAT mice vs control by direct measurements. It is known that increased levels of these SCFAs can stimulate adipogenesis but absorption into the portal system. Thus, it appeared that while both groups of mice were taking in the same amount of calories, the STAT treated mice had an altered microbiome that was extracting more energy from the same input. Validating this hypothesis, the fecal pellets of the STAT mice indeed showed lesser caloric value.
Now, the increased energy efficiency can be accounted for by multiple means:
1) Increased absorption of SCFAs from the colonic epithelium
2) Increased production of SCFAs directly altering the colonic metabolism
2) Increase in the efficiency of storage and conversion of these SCFAs into adipocytes
The authors fail to test the first hypothesis even as they show by gene expression profiling that STAT induced intestinal changes result in substantial changes in the regulation of lipid, cholesterol and triglyceride metabolism in the liver. Upon examining the third possibility, the authors find that the adipose tissue in the STAT mice shows no substantial physiological difference compared to the controls in cell density, local inflammation, or metabolic potential.
The authors thus postulate that the sub therapeutic antibiotic treatment somehow selected for an altered microbiota that was able to extract a higher proportion of calories from dietary complex carbohydrates that were relatively indigestible in the control mice. The resultant SCFAs from microbial activity could then enable enhanced lipogenesis by being absorbed and transported through the portal system.
One thing that does not become clear to my mind is how and why the STAT treatment alters the microbial population. The authors propose that the STAT actually represents a compounded perturbation and thus has profound, long term alterations in the community state. But this does not give a selection pressure favoring the rise of firmicutes vs the bacteroidetes.
A News and Views column in the same issue of nature by Harry J Flint from the University of Aberdeen raises some more interesting possibile explanations for the observed phenomena. Dr Flint proposes that antibiotic treatment may also affect the energy provision for the host - such as by altering the transit of food through the gut which is in turn known to affect SCFA absorption and fiber degradation. He hypothesizes that the microbial signals could differentially influence the muscles that control transit of food through the gut. He also raises the possibility that microbial populations (or their metabolic products) could alter early adipogenesis in the early stages of mammalian growth. Another innovative possibility suggested by Dr Flint is that the antibiotics might suppress bacterial in the small intestine (instead of in the colon where the paper tested) and thus make more digestible nutrients available for absorption in the small intestine.
All these possibilities are all quite intriguing and theoretically plausible (though improbable to me) and one can only say that further experimentation is required for us to completely understand how sub-therapeutic doses of antibiotics affect animal health.
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