The Nose Knows How to Keep Staph at Bay

By: Helen Beilinson

A year and a half ago, I had temperatures over 100°F, could barely concentrate, and couldn’t sleep for more than two hours without waking up covered in sweat. After three days of somewhat tolerating this out of body experience, I realized that this wasn’t some kind of horrible cold and went to the student health care center. I had pyelonephritis, a bacterial infection of the kidney. I spent the next week in the hospital being pumped with antibiotics. Scarily, the antibiotics I was initially given did nothing to the invasive E. coli trying to take over my body. They were resistant to the drug that was supposed to kill them. It took two days of lab tests to realize this fact, after which I was immediately switched to another antibiotic, to which my new bacterial friends were not resistant. Thankfully, I was infection free and out of the hospital soon after.

I was fortunate in that a simple switch to another antimicrobial cleared my unwelcome squatters. In many cases, infectious bacteria are resistant to multiple drugs, termed multi-drug resistant organisms (MDRO), or, more comic book-ly, ‘super-bugs’. Bacteria evolve very quickly; modifying their genomes to select what works best for their environment or even obtaining bits of DNA from other bacteria that would aid them in survival. These bits of DNA encode genes that mutate at some of the fastest rates and that are one of the most frequently transferred from one bacterium to the next. They also encode genes that induce resistance to antimicrobials. The longer certain antimicrobials are used in the clinic, the more bacteria are able to acquire resistance genes, spreading resistance from bacterium to bacterium, from species to species. The need for new antimicrobials has been terrifyingly high for the last few decades because there has not been a new class of antibiotics discovered for the treatment of bacterial infection since 1987. 
Many studies have been aimed at synthesizing new biomolecules in the lab in attempts to find a molecule never before encountered by bacteria that has antimicrobial potential. Other scientists have turned to a surprising place to find naturally occurring antimicrobials: bacteria.

Bacteria occupy what are called niches, a location that has a particular bacterium’s perfect temperature, humidity, and food. Although there are millions of niches that bacteria occupy, from lava pits to our pets’ guts, bacteria still have to compete with other bacteria for their niches. This is particularly true of locations that are not rich in nutrients—the bacteria not only have to fight for space, but also for food. To fight against invaders, bacteria use many strategies, including producing their own antimicrobials. One species of bacteria will produce molecules that harm other bacteria, while the producing species is unaffected by those molecules. These microbially-derived antimicrobial molecules have been the subjects of many scientific treasure hunts. Many niches have been explored, from the ground to the ocean (both of which have been very fruitful locations), but one recently published paper from the University of Tübingen looked for antimicrobials much closer… in their noses.

Any location on the human body that is exposed to the environment has a milieu of microbial life living in it. The nose is no exception. The nose, as well as the upper airway to which it is connected, is very nutrient poor, meaning that any bacteria that live there are in strong competition for space and food. Bacterial species from the human microbiota have been shown to produce bacteriocins, which are antimicrobial molecules. The authors of this study explored nasal commensals in attempts to identify antimicrobials capable of acting on Staphylococcus aureus, a bug that lives in the nose, respiratory tract, and on the skin. Although approximately 30% of the human population has S. aureus living in or on them, during times of immune suppression, S. aureus can cause skin and blood infections. Without antibiotic treatment, S. aureus bacteremia (bloodstream infection) has a fatality rate that ranges from 15 to 50 percent. Unfortunately, the prevalence of S. aureus in antimicrobial filled hospitals has lead the species to be highly resistant to many drugs. For example, MRSA is methicillin-resistant S. aureus, a highly difficult bacterium to treat. Many S. aureus strains are now classified as MDRO, as they are resistant to more drugs than just methicillin. Without novel antimicrobials to treat S. aureus infections, or any other MDRO infection, the fatality rates will only increase.

S. aureus is in the genus Staphylococcus and has many other genus family members, many of whom also live in the human nose. The authors of this study used a previously described collection of nasal Staphylococcus to screen the species’ ability to inhibit the growth of S. aureus. They identified one strain, called S. lugdunensis IVK28, which very strongly prevented the growth of S. aureus. In the hopes of identifying the specific molecule, or molecules, that S. lugdunensis uses as an antimicrobial against S. aureus, which could subsequently be used as clinical antimicrobials, the authors made a mutant library of S. lugdenensis. In essence, this means that they modified the entirety of the genome of S. lugdenensis randomly in many different ways. If a gene is mutated that is critical to repressing S. aureus growth, then that mutant would be unable prevent S. aureus growth. Then, they tested to see if any of the mutants were unable to repress S. aureus growth. When they found one mutant that did not have this antimicrobial property, they sequenced the bacteria’s genome to identify what gene was mutated. They found that the previously uncharacterized gene, which they named lugdunin, was mutated in this line of S. lugdunensis. This is the first clue showing that lugdunin could be a novel antimicrobial.

To test if lugdunin has antimicrobial properties outside of the context of S. lugdunensis, the authors isolated lugdunin on its own and found that it was able to independently act as an antimicrobial on S. aureus growth. This fact is important in that many molecules, particularly proteins, sometimes don’t function alone, working in conjunction with other proteins, or other biomolecules such as lipids or nucleic acids. An independently functioning molecule is much easier to work with, both in basic science characterization studies, as well as in the clinic. Lugdunin is a strong antimicrobial, with the ability to act against various strains of drug-resistant S. aureus and drug-resistant members of the Enterococcus genus, as well as many other bacteria. Importantly, lugdunin did not cause any damage to human cells (important when trying to develop a drug for human use). When the authors used a mouse skin infection model using S. aureus, lugdunin was able to eliminate most or all of the infection, a first critical experiment in demonstrating its potential as a clinically available antimicrobial.
The question of why some people are carriers of S. aureus, while others may go their entire lives without have one such bacterium ever live in or on them, has remained largely unanswered by scientists. To explore whether the presence of S. lugdunensis affects the presence of S. aureus, 187 patients’ nasal swabs were analyzed. A third of the patients had S. aureus, a number close to the national average, whereas a tenth of the patients had S. lugdunensis. The presence of S. lugdunensis, however, strongly decreased the likelihood that the patient had S. aureus. Although it couldn’t definitively be proved in humans, as human testing is strictly frowned upon by the higher powers that be in the scientific world, this finding was a critical step in understanding the relationship between the two Staphylococcus strains.

To further test this antagonized relationship, the authors asked whether lugdunin gave S. lugdunensis the capacity to outcompete S. aureus for nasal space. They found that this, in fact, is true. When they plated both bacteria on agar plates (basically thick jello with tons of nutrients), they found that S. lugdunensis always took over the plates within 72 hours. Even when the plate started as 90% aureus and 10% lugdunensis, three days later, there wasn’t a S. aureus bacterium to be found. When a mutated S. lugdunensis was used, a variant that lacked the lugdunin, S. aureus was able to take over the plate with ease. These findings show that S. lugdunensis is not just a member of many people’s nasal microbiota, but its ability to compete with S. aureus, thanks to its lugdunin molecule, can keep aureus at bay and prevent any potential infections it would cause.

The discovery of a potent antimicrobial that can act on drug-resistant bacteria is important. Of course, there is always the risk that bacteria will develop a resistance to this new antimicrobial, but when the authors of this study tested to see whether they can ‘force’ S. aureus to become lugdunin-resistant, they found that the rate of resistance development was minimal. Whereas S. aureus developed resistance to other drugs after even just a few days, lugdunin resistance wasn’t observed, even after a month. Lugdunin is an exciting new antimicrobial that hopefully will be able to treated MDRO-infected individuals soon. Additionally, as S. lugdunensis is a known safe nasal commensal, a fascinating potential of these findings is infection prevention, instead of treatment. Patients who are at a high risk for S. aureus infection can be colonized with S. lugdunenesis to make the bacteria work for us in exchange for the delicious mucus they feast on. The presence of this S. aureus fighter will lower the risk for S. aureus presence, even already drug-resistant S. aureus, in the nasal cavity, lowering the chance of a life threatening infection. Although it was a quiet field for a while, antimicrobial discovery has only been speeding up in the last few years. Exciting new discoveries are being published every few weeks and our ability to treat infections, as well as preventing them in the first place, is only getting better. Who knew that sharing boogers could save lives?

 

Timing Decomposition with Microbes

By: Helen Beilinson

The last decade has seen many new discoveries that have revolutionized science. Arguably, one of the most influential of these advances has been the appreciation of the impact of microorganisms on human health. In particular, the important roles played by the bacteria, viruses, and other bugs (collectively called the microbiota) that live in or on us, continue to be enumerated. Numerous elegant studies have characterized changes that happen in a person’s microbiota throughout the course of a regular year, during the progression of an infection, or even how a space environment can affect the composition of bacteria in our gut. Recently, a group from the University of California, San Diego explored the changes in bacterial composition during a different phase of life—corpse decomposition.

It might sound a bit gruesome, but the decay of once living things is critical for the cycling of nutrients on earth. The completion of this task requires an extensive arsenal of microbial and biochemical activity. Previous studies had shown that decomposition occurs in a somewhat predictable, stepwise fashion. It was also known that bacteria and other microorganisms are critical for this natural process to occur properly. However, the details of this process were not well understood. The authors of this specific study wanted to know if the environment an organism inhabits dictates the microbial decomposers, whether these microbes come from the host or the environment, and how a decomposed organism changes the environment around it.

To answer these questions, the authors determined the composition of the communities of microorganisms in decaying mice and humans in various environments. Using human cadavers might sound a bit grisly, but it’s important. Mice are good models of various human diseases and are great tools to study many aspects of biology and organismal biochemistry. However, human and mice are still two different organisms, and human subjects were required in this study to verify that their findings in mice matched what occurs in humans. The use of human cadavers is important for the implications and potential applications of this study, as it may be the newest tool in forensic science... but I’m getting ahead of myself.

To identify the families that make up the microbial communities in their samples, the authors utilized a precise technique, 16S rRNA sequencing. This technique takes advantage of the fact that there are some genes that are pretty similar in bacteria that are closely related, and grow more different the less related they are. By sequencing all the microbes, the authors are able to group them into their families and compare how similar or different the microbial populations are between different experimental groups.

An exciting preliminary piece of evidence these authors observed is that the previously described stages of decomposition followed hand in hand with a very precise and dynamic microbial community. The microbes present on day 1 are different from those that emerge on day 4 which again are different from those on day 10. At every stage, between day 1 and day 71, the microbial communities were unique. Perhaps surprisingly, when the authors changed the location of where their mouse specimen was decomposing, there was no effect on the microbial decomposers! A microbial community from a mouse decomposing in a desert environment on day 7 was almost identical to that from a mouse decomposing in a forest on day 7. Seasons also did not significantly impact microbial populations. These same data were obtained using human cadavers.

Based on the latter piece of information, one might expect that if microbial decomposers are more or less the same in different environments, which these decomposers would come from within the host itself. However, the authors found that the soil is the primary source of the microbes, even if the soil type and environment is different. It’s important to note that 16S rRNA sequencing is not the best technique for identifying specific microbes. It is mostly used to identify families of microbes that are closely related. Families of microbes tend to have similar functions, meaning they can carry out similar reactions. These data together imply that because specific microbes carry out specific reactions and that there is a predictable change in microbes over the course of decomposition, one could predict that the biochemical changes carried out by microbes can be tracked in sequential steps during the course of decomposition.

To further explore this question, the authors examined biochemical reactions taking place in the abdomen of the decaying specimen. They found that indeed, throughout the process of decomposition, there are specific reactions that can be detected at each step. The biochemical reactions that take place correlate almost perfectly with the presence of particular microbes that can carry out this process. Interestingly, the authors were also able to show that the soil around decomposing organisms also has such post-death dating properties, in that the products of the biochemical reactions occurring in the organism seep into the soil surround it, changing its chemical properties. The products produced, such as nitrate and ammonium, are used by plants to grow. Although it is a tad ghastly to think about, mammalian decomposition is important for the cycle of life on Earth. The products of decomposition allow for plants to grow which feed the living mammals.

Although now a fairly simple technology, sequencing of the microbiota of an organism has been an incredibly powerful tool in the biomedical sciences. This study has shown that another, perhaps surprising, field that may benefit from this technology is forensic science. Although there are technologies in place to help forensic scientists identify when a person has died, these are often not very precise. This paper’s methods were able to distinguish time of death with a one to two day accuracy based on the microbes found in that body and the biochemical reactions occurring. The microbes around us clearly have a constant influence on our lives: from our birth to our death, microorganisms make us who we are.

Why don’t we kill our microbiota?

By: Zuri Sullivan

If you didn’t already know that humans are colonized by trillions of bacteria, that these bacteria make up much more of our bodies than our own cells, and that they play an important role in our physiology, health, and disease, then you probably didn’t read Helen’s piece about the microbiota and defense against viruses. The “microbiota” refers to the collection of microorganisms that live in and on our bodies. A search for “microbiota” returns close to 14,000 academic papers. To call this a current hot topic would be a massive understatement. Why are scientists so excited about the microbiota? First, major technological advances have allowed scientists to begin unraveling the complex community of bacteria, fungi, and viruses that colonize our bodies. Additionally, mounting evidence suggests that the microbiota can influence nearly every aspect of our physiology, which raises important questions about its interactions with other cells and tissues in our bodies. 

There’s a lot to be learned about the microbiota and its functions, but one of the biggest unanswered questions in the field is why all of these bacteria are overlooked by our immune system. Most of us know the immune system as the mechanism by which our body eliminates disease-causing agents, or pathogens. These pathogens are bacteria, viruses, and fungi—the same organisms that make up the microbiota. One of the biggest unanswered questions regarding the microbiota is why our immune system doesn’t eliminate our own microbial population, but does eliminate those microbes that are pathogenic. The initiation of an immune response against an invading species relies on the detection of pathogen associated molecular patterns (PAMPs). PAMPs are molecular structures that are present in microorganisms, but not in host cells. For example, a particular sugar-like molecule, lipopolysaccharide (LPS) is present on the surface of some bacteria, but absent from mammalian cells. Thus, by targeting attacks against cells that have LPS, the immune system can efficiently target a wide array of bacteria without risking damage to host cells. In addition to being distinctly non-mammalian, another critical feature of PAMPs is that they are conserved amongst microbes. This means that PAMPs tend to be molecules that serve critical functions for microbial survival, and thus they are seen in an array of microbial species, including pathogens (microbes that make us sick) and commensals (microbes that have neutral or beneficial effects on our health). This raises an important conundrum: if the immune system recognizes PAMPs that are common to all microbes, why don’t we attack commensals?

A paper published last week in Science provides an exciting clue to how our commensal bacteria survive some of the host’s innate defenses. They looked at antimicrobial peptides, which are chemicals made by immune cells that can directly kill bacteria. They’re like antibiotics made by your own body, and they are an important innate defense mechanism against bacterial infection. The team of researchers asked whether commensal bacteria might be resistant to antimicrobial peptides, which would allow them to survive in the host. Their hypothesis was that commensal bacteria that are adapted to live in our bodies might have evolved mechanisms to tolerate antimicrobial peptides, while pathogens have not.

They found that a common commensal species, Bacteroidetes thetaiotamicron  (B. theta) was more resistant to a particular antimicrobial peptide than pathogenic bacteria. Next, they identified the B. theta gene that was responsible for antimicrobial peptide resistance, a gene called LpxF. They then asked what role this gene played in the ability of this commensal organism to survive the host immune response to a pathogenic bacteria.

This was an important question, because it illustrates an important principle about how the microbiota helps us protect ourselves from pathogenic bacteria. When we’re infected with an intestinal pathogen, gut immune cells detect the infection and induce many defense mechanisms, one of which is the secretion of antimicrobial peptides. These antimicrobial peptides need to be able to target the pathogen, while sparing the commensals that provides other benefits to the host. Thus, by having a gene that confers resistance to antimicrobial peptides, the commensal has an advantage over the pathogen in the context of an immune response.

This was borne out in an experiment wherein the researchers added two different bacterial strains to the intestines of germ-free mice (mice who are raised to not have their own microbiota): wild-type (normal) B. theta or B. theta that were lacking LpxF (a mutant strain), and therefore lacked resistance to antimicrobial peptides. They then infected these mice with a known pathogen, Citerobacter rodentium. They found that while the LpxF mutant strain of B. theta was outcompeted by the pathogen, the wild-type B. theta survived the immune response.  

The findings of this study illustrate two important principles. First, they provide a mechanism that explains how a commensal bacterium survives a host defense mechanism that is directed against all bacteria. Second, they show that this survival mechanism helps commensal microbes compete with pathogens for colonization of the gut. These principles are relevant to both the continued understanding of the role of the microbiota in host defense, and in understanding the co-evolution of the immune system and commensal microbes. This co-evolutionary relationship, whereby our evolved mechanisms for eliminating bacteria are resisted by commensals that have evolved to live in our bodies, is important for the continued study of the microbiota and its impacts on our health. 

I Kid You Not, A Disease Exists That Makes You Drunk, Constantly

By: Ross Federman

A bar I used to frequent serves a delicious frozen drink known as a “Constant Buzz.”  It sounds like an appealing concept, a nice basal level of ongoing intoxication, but did you know that in some incredibly rare cases, individuals find themselves in this situation without any choice?  Auto-Brewery Syndrome (or gut fermentation syndrome) is a disease whereby patients literally brew alcohol in their guts.  Imagine not taking a single sip of beer, wine, or liquor, yet still constantly registering above the legal limit on any manner of blood alcohol or breathalyzer tests.  This is the unfortunate case for those rare few that find themselves with this disorder.

How does it happen?  It was a mystery for years.  In fact, it’s likely that some may have been incarcerated, fired from jobs, suspended from school, you name it, all because they were drunk against their will with no explanation to offer as to their erratic behavior.  Recently, we have gained significant insight into the nature of our gut microbiota and the profound role that it plays in human health.  The microbiota is usually discussed in terms of the species of bacteria that comprise it; yet other microorganisms are present, as well.  In most healthy microbiota, a small population of the commonplace fungal yeast species Saccharomyces cerevisiae lives in your gut with all the other microorganisms.  S. cerevisiae is also known as “Baker’s Yeast” and is the species often simply referred to as “yeast” in the majority of baking and beer brewing applications. You and everyone around you likely have some S. cerevisiae hanging around in your guts, and the amount of it is largely kept in check by your immune system, other microorganism species, and competition for nutrients.  However, in some rare cases, this population is not kept to the proper minimal levels and grows wildly out of control. 

At the heart of this phenomenon is the fermentation process.  It is found in both yeast and bacteria, though the specific fermentation products differ.  In both cases, sugars or carbohydrates are broken down to carbon dioxide in order for the microbes to produce energy in an environment lacking oxygen (such as our guts).  For bacteria, lactic acid is the second byproduct along with carbon dioxide, but in yeast, ethanol is produced in lieu of lactic acid.  Thus by increasing the ratio of S. cerevisiae to bacterial species in the gut, greater amounts of ethanol are produced, and past a certain threshold, the ethanol metabolic product seems to reach levels where it is in a great enough concentration to equate to imbibing ethanol in the form of an alcoholic beverage.  It should also be noted that this same fermentation process is used to achieve the alcohol content in said beverages, though this, of course, does not take place directly in our stomachs.

Even with a greater understanding of the microbiota, the precise nature of why these yeast populations can grow so large and lead to ongoing intoxication remains unknown. Cases are so rare that really everything we know about this disease is almost entirely anecdotal.  Perhaps the patients ate an absurd amount of sourdough after taking antibiotics that would have wiped out many of the bacteria just prior to yeast colonization?  Whatever oddball circumstance lead to this, the end result?  Enough yeast in your gut to lead to fermentation levels that are actually sufficient enough to influence blood alcohol levels.  Sounds pretty awesome if it only lasts for the three days you spend at Coachella, but week in and week out, it would probably get old pretty quickly.

Viral infections and the microbiota

By: Helen Beilinson

Take home points:

1)    The microbiota can influence viral infections—exacerbating disease or preventing disease—so we need to be mindful of how we take antibiotics during viral infection

2)    Antibiotic treatment during flu infection can exacerbate disease

3)    Antibiotic treatment during other viral infections could prevent viruses from taking advantage of immunosuppressive properties of microbiota

 The microbiota—two words that have been splattered across scientific papers, the evening news, and “yogurt for women” packages. The multitude of bacterium that live within and on us constitutes, essentially, another organ that plays a role in many aspects of development and maintenance of the larger organism. It has been long appreciated that the microbiota plays a beneficial role in a host’s metabolism. However, only recently it has been shown that the microbiota is a significant player in the host’s immune system. In fact, it has a huge influence on how we interact with external pathogens and on the pathogenesis (the development of disease) of these pathogens. The predominantly bacterial microbiota live mostly at mucosal surfaces of the host (such as the gastrointestinal tract, genital mucosa, and upper airways), as well as on the skin. Interestingly, viruses are predominantly spread through routes where they would interact with the aforementioned microbiota. Thus, it is highly probable that the microbiota and viral pathogens interact and influence each other in some way. In fact, it has been documented that the microbiota can either obstruct or advance infections of various viral pathogens (or have no effect).

In mouse studies, it has been shown that eliminating the microbiota, either through antibiotics treatment (treatments against bacteria) or by raising mice germ-free (meaning that they have never been colonized with any bacteria), is very detrimental during influenza infection. Mice have more viral particles, higher mortality rates, and more degeneration and death of their lung tissue without their proper bacterial population. The mechanism of microbiota-dependent protection currently is unknown. This trend is incredibly important to study for its implications in human health. Flu infection changes the landscape of our lungs, making it easier for bacteria to infect the lungs. One such bacterium is Streptococcus pneumoniae, a causative agent of pneumonia. The most fatal cases of influenza are due to such secondary bacterial infections. Increases in mortality due to co-infection have been confirmed in mouse models. When patients enter the hospital with bacterial pneumonia, they are placed on antibiotics. This could potentially exacerbate flu disease by eliminating the beneficial bacteria of the patient’s microbiota, leading to death. Unfortunately, there have not yet been studies to show that antibiotic treatment causes patients to fare worse in co-infection situations.

In theory, our immune systems are activated once they sense an invader via certain molecular patterns that are unique to the invaders. For all intents and purposes, however, the molecular patterns decorating pathogenic bacteria are the same as those decorating the bacteria composing our microbiota. Although one of the current mysteries of the microbiota is why our immune system doesn’t eliminate it, it is known that these commensal organisms possess immunosuppressive properties. For example, a molecule that decorates the surface of particular bacteria is called lipopolysaccharide (LPS). There are various forms of LPS, some forms are immunostimulatory, while others, such as those expressed by the microbiota, are immunosuppressive. It has been shown that there are viruses that take advantage of the immunosuppressive properties of commensal LPS to dampen the responses of the host, allowing for active viral replication. Thus, although depletion of a host’s microbiota may be detrimental in flu infection, in cases where the microbiota aids viral replication and disease progression, antibiotics may be a source of treatment.

Studies in mice utilizing two different retroviruses (the family of viruses to which HIV belongs) have shown that these viruses require the presence of the microbiota to be present to flourish. By binding to LPS of the gut bacteria, the viral particles stimulate immunosuppressive pathways, allowing the virus to replicate without the host attempting to eliminate it. These studies were done in mice with murine viruses, so the question remains: Do human viruses interact similarly with LPS? Potentially.

It has been shown that HIV virions can directly bind to LPS molecules. It has also been shown that the presence of LPS during HIV infection (in a series of experiments in cells, not full organisms) decreases the “activated” state of antiviral immune cells, called plasmacytoid dendritic cells, preventing them from potently attacking infected cells. LPS is recognized by Toll-like receptor 4 (TLR4) in our immune system. In an unknown fashion, different LPS variants stimulate TLR4 to produce either a pro- or anti-inflammatory state, which is defined by immune signaling molecules called cytokines. An HIV protein, Tat, has been shown to stimulate the same immunosuppressive pathway as the mouse viruses by interacting with TLR4. This study did not show whether or not LPS was present in the experiments shown (LPS is a pesky molecule that is incredibly difficult to eliminate in a lab setting). Either way, it points to interesting evidence that HIV, as the mouse retroviruses, has possibly evolved the ability to take advantage of the immunosuppressive properties of the microbiota to allow for its replication. Thus, antibiotic treatment could be a potential treatment for HIV replication by decreasing the amount of commensal-expressed LPS that the virus may take advantage of to evade the immune system.

Living predominantly in areas that are directly exposed to the elements and invading pathogens, our microbiota may hold essential information regarding how our body battles infection and either wins or loses. The modulation of our live-in bugs will certainly be a future step in medicine in aiding the treatment of various infections, including those of viral pathogens. So next time you have a viral infection, be sure to ask your doctor whether it is safe for you to take antibiotics—or whether it could possibly help your immune system eliminate the infection.

It is important to note that neither the flu nor the HIV findings have been confirmed in humans, particularly the HIV studies. The intricacies of the microbiota, and particularly the details of how it interacts with intruding viruses, are still very enigmatic. Presented here are very brief summaries of very few papers on this topic, these should not be taken as medical advice.