A discussion of the gut microbiota composition, classification of bacteria, as well as the use of antibiotics.
Written by: Akshaya, Kavya, and Haritha
Antibiotics have been shown to cause ‘microbial dysbiosis’ which is the disruption of gut microbiota.
What are the main players in the human gut microbiota?
The gut microbiota is made up of bacteria, archaea, and eukarya which end up in the gastrointestinal (GI) tract of the host (1). It is estimated that there are more than 10^14 microorganisms in the GI tract, which, as we have mentioned in the previous blog post, largely surpasses the amount of human cells (1) (though recent research suggests that the amount of bacterial cells are almost equal to the amount of human cells). At the beginning of the growth stages of the individual after birth where the microbiome is not as diverse yet, Actinobacteria and Proteobacteria are the two phyla which can be found in the host. By the time the host is older than 65, the phyla of Bacteroidetes increases, as well as Clostridium cluster IV (Clostridia are bacteria which are gram-positive, which we delve into later in this post) (1). Research has found that the ratio of these bacteria (mainly Bacteroidetes and Firmicutes) in the human gut influences whether one is obese or lean, which is affected by the different communities of bacteria in the gut (8). The GI tract is one of the largest interfaces between the host, environmental factors, and antigens in the body (1).
We have discussed phylogenetic trees and their purpose in our post on Part 1 of Microbiota, but to refresh, these diagrams can help us understand the microbiome better (refer to the diagram below). The data analyzed on phylogenetic trees can allow for unclassified microorganisms to be categorized, as well as to understand the evolution of microbial communities by testing hypotheses of various similarities and associations of traits (4).
A study of 2,172 identified species which were isolated from humans were organized into 12 phyla. Of these phyla, 93.5% were part of Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes (1). We have 386 identified species in our microbiome which are anaerobic (meaning there is an absence of free oxygen) and so, are found in mucosal parts of our bodies (1). Since there is no available oxygen in our large intestine and colon, these bacteria survive without the presence of oxygen.
There are four phyla of bacteria that we will delve into now, which mainly make up the gut microbiota. Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria are most commonly prevalent in our gut. Although most of the species of these bacteria have beneficial effects on the host, there are some that are also pathogenic. Let’s start with Bacteroidetes. Let’s start with Bacteroidetes.
Bacteroidetes:
This genus includes nonpathogenic commensals (as mentioned in the previous post, commensal refers to an organism which lives inside and benefits from another organism, but does not harm or benefit the host). The relationship between the host and these microbes are now characterized by mutualism, meaning both the host and the Bacteroidetes benefit from the relationship. Bacteroidetes exist in the GI tract of humans and also make up most of the feces, but they are also present in ecological niches such as soil or the ocean, working to degrade polymeric (complex) organic matter (6). As of a census of phylogenetic divisions created in 2010, Bacteroidetes have been categorized in four different classes and make up 7,000 different species (6).
The function of the Gut Bacteroidetes is mainly to degrade the biopolymers in the large intestine, though they help maintain a healthy gut by producing butyrate, “an end product of colonic fermentation,” and aid in bile acid metabolism as well as “transformation of toxic and/or mutagenic compounds” (6).
Firmicutes:
With Bacteroidetes, the Firmicutes phyla make up the large majority of the microbes in our GI tract. Firmicutes consist of the second most plentiful bacterial phylum (7). Firmicutes are also commensal bacteria, anaerobes, and can be found outside the GI tract of animals. They are used in processes such as fermentation of wine and beer, the transformation of yogurt from milk, as well as bioremediation, which is the process where living organisms are used in removing contaminants /toxins from water, soil, etc. (8).
In the gut microbiome of humans, Firmicutes provide betterdigestion of fat, which is necessary for energy, but they are also linked to higher risks of obesity (8).This is the reason why some people gain weight even with dieting and more exercise (8). The gut microbiota composition heavily influences the metabolism of the host.
Actinobacteria:
Actinobacteria are another one of the largest phyla with lineages found in domain Bacteria (11). They are gram-positive and mainly free-living organisms which are present in marine ecosystems as well as terrestrial (11). They have different functions in their relationships with higher organisms as they have adapted to different lifestyles and some members are pathogens. Unlike Bacteroidetes and Firmicutes, Actinobacteria are aerobic, but they can also be heterotrophic. Heterotrophic means that they cannot make their own food from photosynthesis, but get it from other organic substances, and chemoheterotrophic, meaning they use inorganic energy sources in order to make organic compounds from CO2 (carbon dioxide).
Although Actinobacteria have interesting relationships with protozoans, plants, and beetles, they also play a part in our gut. Actinobacteria, along with Firmicutes, were found in higher numbers in those who didn’t have diabetes, compared to those with diabetes, but pathogens can also cause various human and animal infections (11).
Proteobacteria:
These are gram-negative bacteria (explained below). They make up pathogenic species which includes Salmonella (5). Another phyla called Myxobacteria and Proteobacteria produce secondary metabolites which are Actinobacteria (5). These metabolites “target cellular structures that are rarely targets of other compounds…” (5). The function of Proteobacteria is to aid in healthy gut function by preparing the gut for anaerobes (12).
Proteobacteria are the most diverse bacterial phylum which can be found in fecal microbiota of healthy animals, but also are pathogens which can impact the health of the host. Additionally, they can cause metabolic and inflammatory disorders if they are present in high amounts (12).
What is the difference between Gram + / Gram - bacteria?
There are many ways to characterize bacteria. From the cell shape to their metabolic preferences, classifying bacteria helps create a better understanding of their function and potential medications to treat them. A common and important differentiation is made between gram-positive and gram-negative bacteria. You might think, there are ‘positive’ and ‘negative’ bacteria? This distinction does not correlate with whether they have positive or negative effects on our bodies nor does it have to do with their ‘attitude’. In fact, gram-positive refers to bacteria with a thick cell wall as the outer layer whereas gram-negative bacteria lack the thick cell wall (10). It is important to note that gram-negative bacteria do have this wall, but it is thin and is covered by an outer membrane made of lipopolysaccharide, which consists of carbohydrate, lipid and protein. Now let’s take a look at the cell wall.
The cell wall is made out of peptidoglycan (“Pep-ti-do-glycan”). The peptidoglycan complex forms a sturdy barrier, protecting the bacterial cell. Glycan refers to the glycosidic bonding between two molecules. And these glycosidic bonds are present in between carbohydrate units called monosaccharides. In other words, monosaccharides (which are the single units of carbs/sugars) link together by glycosidic bonds to form complex structures. In fact, carbohydrates have very important roles in forming structures (cell walls) as well as serving as biomarkers, helping to distinguish between our own cells and foreign cells. Refer to the diagram below for a visual representation.
Peptidoglycan is a carbohydrate structure that forms repeating units of the cell wall in bacterial cells (10). The peptidoglycan cell wall is a target of some antibiotics. So where do the names gram-positive and gram-negative come from? To determine whether a bacteria is gram-positive or gram-negative, a gram staining procedure is followed. As the name of the procedure implies, it involves the use of a stain known as crystal violet which is purple in color (9). Let’s try and run through the experiment. Say we have a gram-positive and gram-negative bacteria. Our task is to determine whether it is gram-positive or gram-negative using the gram stain procedure. First, we add the bacteria to a microscopic slide. We start by adding the crystal violet dye to both of the bacterial slides. The purple dye adheres to the peptidoglycan cell wall, appearing purple. Then we add iodine to each of the slides, forming an iodine-crystal violet complex. The next step is what reveals whether the bacteria is gram-positive or gram-negative. When we add ethanol to the slide, some of the dye is washed away. However, since the peptidoglycan layer of the gram-positive bacteria is thick, most of the dye is not washed off. On the other hand, the thin peptidoglycan layer of the gram-negative bacteria easily allows for the dye to be washed off. Then to make the gram-negative bacteria much more visible under the microscope, we use another dye called Safranin which is red in color (this is also known as counterstaining). We add Safranin to both the slides. The outer membrane of the gram-negative bacteria will be dyed red. The cell wall of the gram-positive bacteria is still saturated with the purple dye and is not dyed red (9). When we look under the microscope, we are able to easily distinguish which is gram-positive and which is gram-negative. Gram-positive will appear purple, gram-negative will appear pink/red.
Gram staining is one such staining procedure that allows for a quick analysis of what type of bacteria we have at hand. Likewise, there are other staining procedures that help characterize the bacteria. You may be wondering, why are the gram stain results important? Read on to the antibiotics section to find out.
How are antibiotics involved?
The type of antibiotic used depends on whether the bacteria is gram-positive or gram-negative. Peptidoglycan makes up the bacterial cell wall. In gram-positive bacteria, this peptidoglycan layer is the outermost layer. In gram-negative bacteria, the peptidoglycan layer is surrounded by lipids on the outside. As a result, antibiotics that target the peptidoglycan layer will only work on gram-positive bacteria since this layer is on the outside. These same antibiotics will be ineffective on gram-negative bacteria. For example, beta-lactam antibiotics such as penicillin, target the outermost peptidoglycan layer. Peptidoglycan reacts with an enzyme called transpeptidase to form an intermediate structure. This intermediate structure then reacts with another peptidoglycan molecule to form ‘cross-links’. However, when a beta-lactam antibiotic reacts with the peptidoglycan cell wall, it prevents cross-links from forming. In simple terms, penicillin affects the proper formation of the peptidoglycan cell wall. This causes the bacterial cell wall to burst and the bacteria dies.
Antibiotics have been shown to cause ‘microbial dysbiosis’ which is the disruption of gut microbiota. This has been shown to cause several diseases such as diabetes, obesity, asthma, and inflammatory bowel disease among others (3). Since antibiotics are broad-spectrum, or act on many different kinds of bacteria, several commensal microbes are also killed or inhibited in the process (3). Short chain fatty acids (SFCAs), which are produced by bacteria during the fermentation of fibers, play an important role in maintaining the integrity of the intestinal epithelium (layer of cells) and preventing gut inflammation. When these essential bacteria are depleted, the production of SFCAs declines and results in gut inflammation (3).
What is antibiotic resistance?
Antibiotic resistance occurs due to the overuse of antimicrobial agents such as antibiotics and antifungals. For example, certain strains of bacteria have a gene for resistance to a particular antibiotic. When all other strains of the bacterium are killed by the antibiotic, this resistant strain is the only one that remains. Now, this strain begins proliferating and renders the antibiotic ineffective (2).
Clostridium difficile is a spore-forming bacteria that is gram-positive and anaerobic. It cannot thrive in competitive environments. When antibiotics kill all other gut bacteria, C.difficile spores are then able to grow and thrive in the gut (3). This bacterium is known to cause antibiotic associated diarrhea. C.difficile outbreaks are a big concern in hospital settings where disinfectants used to kill other bacteria on surfaces do not kill this one, thereby causing outbreaks.
Our gut microbiota is a complex ecosystem of essential bacteria which determine the health of the host. The characterization of these bacteria are vital in order to determine how to treat pathogenic strains. In this blog post, we discussed some of the major phyla in the microbiome to understand their effects in our bodies as well as characterization methods used to classify different microbes. Ultimately, the classification of the gut microbial composition will enable us to target treatments such as the safe and effective use of antibiotics.
Stay Informed, Stay Tuned, Stay SAFE!
Resources:
Medical Microbiology, Kaplan PCAT Prep Plus
https://www.sciencedirect.com/topics/medicine-and-dentistry/proteobacteria
https://www.sciencedirect.com/topics/immunology-and-microbiology/firmicutes
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