Microbiology: Gram-Positive Coccipg. 1
Sonia Nkashama
The audio didn’t start until the middle of slide 4.
4. Also ubiquitous in the human population is S. epidermidis and several others in this group. They are always present on the skin; everyone has this Staphylococcus. Usually it does not cause any problems. But if you get an IV put into your arm for some reason (to give fluids or medications, etc), this organism has a special ability to attach to plastic IV catheters. So it has a tendency to cause bloodstream infections, once you have allowed it to get into the bloodstream by entering with a foreign body. It’s not usually so virulent that it can go through intact skin to get into the bloodstream. But if you put an IV catheter in a vein and allow the organism to get there, it can certainly cause problems. Staphylococcus saprophyticus is an organism that actually causes urinary tract infections in young women. It’s actually found in the urinary tract and can cause infections there.
5. As far as some of the microbiological characteristics of the Staphylococci, there’s one thing that they all have in common: they stain as round, Gram-positive organisms. They will usually appear in grape-like clusters; this is a typical Gram stain. You can look at this Gram stain, and by the fact that it is dark-purple staining, round cells that appear in bunches, it is most likely a Staphylococcal organism. You can’t say which Staphylococcus that it is, however. They are usually a micrometer in size, and they occur in clusters.
They are very easy to grow in a clinical lab. They’ll grow on most of the media that we have; they’ll grow under aerobic conditions. S. aureus is β-hemolytic. We will say more about hemolysis when we talk about the Streptococcus. And you will actually get to see hemolytic patterns in the laboratory. The Staphylococcus produces several exotoxins, and one of the toxins it produces is a hemolysin. This allows it to hemolyse red blood cells. If you grow the organisms on a blood agar plate (usually a sheep blood agar plate—standard media that is used in Micro lab), you’ll see a clearing around the organisms. This is a complete hemolysis, a complete lysis of the cell. This is referred to as β-hemolysis.
Something else that you can use to identify S. aureus is that it will ferment mannitol. If you put the Staphylococcus on a plate that contains mannitol as a carbohydrate substrate, it will ferment the mannitol. And in doing so, it will cause a pH change. The pH indicator in the medium, such as phenol red, will cause the agar plate to turn from red to yellow, thus indicating mannitol fermentation. Two other important characteristics that help you identify S. aureus are the catalase and the coagulase test. You will do those in the lab next week. The catalase test is actually a test for the presence of hydrogen peroxide, and it will separate the Staph’s from the Strep’s. The coagulase test is another test that will separate S. aureus from some of the others. These are very important tests that you need to understand the biochemical basis of.
6. This is the reaction that you see in the catalase test and how you do it. You take hydrogen peroxide, and if you have the catalase enzyme in the Staphylococcus, it will produce water and oxygen, and you will see bubbles. And the way that you do the test is you just add hydrogen peroxide to a colony of the organisms that you are looking for, and you see if you get bubbles. So this is an easy way to distinguish the Staphylococcal organisms from the Streptococcal organisms. The Strep will be catalase-negative, while the Staph will be catalase-positive.
So why would a bacterium want to produce catalase? It’s a virulence factor. Peroxides are toxic to many bacteria. We produce peroxides in our body as part of our innate host immune defense system. Peroxides will break down bacteria. If the bacteria can get ahead of the game and break down the peroxide, the peroxide can’t do anything to the bacteria. So it indeed is a virulence factor that many bacteria possess; Staphylococcus is among those bacteria that do possess this trait.
7. The other reaction that I mentioned is the coagulase reaction. Coagulase is an enzyme that actually converts fibrinogen to fibrin. It will produce a clot around the Staph. And the advantage to this is that in the body, the organism that is producing coagulase will clot plasma around itself. Therefore it will protect itself from complement, antibodies, WBCs, etc. that the body is producing to try and get rid of it. So this is way that it has of protecting itself and avoiding the host immune system. This happens to be a very useful test because it will distinguish S. aureus and the relatively few other staph’s that are coagulase-positive from a large group of staph which are coagulase-negative. This group includes S. epidermidis and many others that are not as important as pathogens. Coagulase again is a virulence factor, and it’s one that we use in the lab all the time to help identify S. aureus and distinguish it from the other Staphylococci.
The classic way that you do the test is the free coagulase test. You take rabbit plasma, put the Staph in the rabbit plasma, and it will clot if it produces coagulase. If the plasma doesn’t clot, then it’s a coagulase-negative Staph. This may take a few hours to produce the reaction, so it’s not as efficient. One of the ways that we do it is to look for clumping factor, which is a surface coagulase test. We just have latex particles that have the fibrinogen and IgG coated on the surface of the particle. The IgG is to protect protein A (another virulence factor). This is a way that you can test it quickly. You can just look quickly to see if there is agglutination of the Staphylococci with the latex particles. And that is presumptive identification of the bound coagulase that is present on the surface of the Staph. So you can get that info very quickly. This will be clearer when you do the test in lab. So remember catalase separates Staph and Strep; coagulase will distinguish S. aureus from the other coagulase-negative Staphylococci.
8-9.The staphylococci have a number of different surface structures that also enable it to be a pathogen. And of course, like most Gram-positive bacteria, it has a large layer of peptidoglycan. It also has teichoic acids & protein A (another surface antigen). This protein is also a virulence factor because it allows the bacteria to bind antibody. It will bind the Fc fragment of IgG and inactivate it. By doing this, it helps protect the Staph from the antibody that the host is producing. Clumping factor is the bound coagulase that I mentioned before. In other words, Staph actually has a surface molecule on it that is the clumping factor expressed, as well as an enzyme that it actually secretes into the environment. Some staphylococci actually have a capsule, a polysaccharide slime layer. This is an antiphagocytic action, helping evade it from the host immune system. And in the case of S. epidermidis, it also has this layer, which is part of what allows it to get through plastic, etc.
The peptidoglycan, a big part of the Staphylococcal cell wall, is also a virulence factor because it will stimulate the host to produce interleukins, inflammatory mediators, as well as opsonic antibodies that will help to bind the Staphylococcus. It’s also one of the things that stimulates neutrophils. Remember that the prototype lesion of Staphylococcal infection is an abscess. S. aureus is one of the most common causes of abscesses anywhere in the body. Part of the reason that you get an abscess is that things that are on the organism are stimulating the WBCs (mainly the PMNs) to come to the place where the Staph is invading the body. Phagocytosis is a very important defense mechanism against this organism because it’s an extracellular organism that gets engulfed by the neutrophil and it will die.
Question: How is the peptidoglycan a virulence factor? It’s a virulence factor in that it stimulates the host immune response. And in stimulating the host immune response, it’s causing damage. Remember part of the thing that we are doing in trying to kill the bacteria, we are producing a lesion. An abscess is disease caused by Staph. That’s a pathologic lesion. The reason that the Staph produces the lesion, to a large degree, is that the peptidoglycan stimulates PMNs to come. So it’s a virulence factor in that it’s allowing the Staph to produce disease to produce an abscess.
It’s also involved in the binding of the bacteria to the tissue. One of the important things that bacteria have to do to cause disease in the body is to attach themselves somewhere. If they are going to produce toxins and cause tissue damage, they have to get into close approximation with the body. So the fibronectin component in the host cells actually is a target of the cell wall peptidoglycan. This is one of the things that actually allows the Staph to attach so that it can then produce its toxin and local damage there. I already mentioned Protein A as binding antibody. And of course, I mentioned the capsule as well.
10. In addition to the cell surface structures that are virulence factors in the Staph, it produces a lot of soluble virulence factors. These are exotoxins, usually enzymes or substances that the Staph produces and secretes around itself. They can actually travel to other parts of the body and cause disease. This is one of the reasons that S. aureus is such a successful pathogen; it has so many different ways that it can cause disease and so many different diseases that it can cause. For example, we talked about the catalase & the coagulase. An enzyme, hyaluronidase, elaborated by the Staph will actually destroy connective tissue. You learned in Microanatomy last year about the fascia and the tissue and what holds the tissues together. Well, hyaluronic acid is one of the things that binds your tissue together. One of the things that the Staph does is produce an enzyme called hyaluronidase; this allows it to spread in the tissues by breaking down the glue that holds your tissues together. This allows it to spread through fascial planes and the cause of disease is spread this way.
S. aureus is resistant to many antibiotics. One example is a β-lactamase that it produces. The β-lactams includes all of the penicillin and cephalosporin antibiotics (some of the most common antibiotics that we use to treat infections). When penicillin was first introduced in the 1940s, all staphylococci were susceptible to it. Within a very short time, most staphylococcus became resistant to penicillin. This is because it will produce an enzyme that breaks down the β-lactam ring and destroys the penicillin. β-lactams work because they have this β-lactam ring in their structure that resembles the amino acids that are used to link the peptidoglycans together. And by breaking down and opening the β-lactam ring, the penicillin molecule will no longer resemble the substrate to cross-link the cell wall binding. Andthat’s why penicillins don’t work on S. aureus. Several other enzymes are also produced by the Staph aid it in producing human disease. I won’t go into detail about all of them; I just mention what they are on this slide.
11. In addition to some of these, you also have cytotoxins and leukocidins. One particular substance called the Panton-Valentine leukocidin is an enzyme that allows the Staph to lyse WBCs. Remember all of these virulence factors not only are the things that allow the Staph to produce lesions, but they’re also the things that allow it to damage the host immune system so that they can survive. WBCs are very important in getting rid of Staph. So if you can kill the WBCs before they kill you, this is an example of what successful bacteria can do. This is a particularly important virulence factor in the new strains referred to as community-acquired methicillin-resistant Staph. They nearly all have this Panton-Valentine leukocidin.
The Staph produces a lot of different enzymes that, when it’s present in the body, can damage tissue. And this is part of the reason that you get liquefactive necrosis when you have an abscess due to this organism. That’s due not only to the enzymes that the PMNs are secreting to try to kill the bacteria, but it’s also from the enzymes that the bacteria is secreting to help spread through the human body. There a lot of these different toxins that aren’t produced by every strain of Staph. So this is why not every Staphylococcal disease is the same. For example, there is one disease referred to as the “Scalded Skin Syndrome” because of a toxin that’s produced by some strains of Staph called the exfoliatin toxin. Literally what it does if the Staph gets into the body or onto the skin and produces this toxin, it interrupts the intercellular skin junctions and causes the skin to literally slough off. It looks like if you had a bad burn, got a blister, and then the skin peeled off. That’s what the skin looks like because of this toxin that the Staph is producing.
Another thing that you see is the toxic shock toxin. In the late 1970s, this was first described as an entity called “Toxic Shock Syndrome.” This syndrome is due to the toxic shock toxin that’s produced by the Staph; this toxin is referred to as a superantigen. It stimulates the T cells to release lots of cytokines and the pro-inflammatory cytokines. Ultimately what you get is endothelial damage to the capillaries in the skin and this is why you get leakage of the capillaries and you get a rash associated with this.
Staph can also cause food-borne disease. Along with the other Gram-(-) bacteria, the enterotoxins that are produced by some strains of Staph are actually known to produce food poisoning because they stimulate vomiting by interaction with the GI neural receptors. So this is where you can actually get a food-borne disease by eating the toxin while eating the bacteria. If you ingest the toxin, as may be found in potato salad or ice cream or other foods that have not been properly maintained or refrigerated, the Staph, if they grow in these foods, can produce the toxin. Then if you ingest the toxin, whether you ingest the bacteria or not, you can get the toxin acting in your body and mainly producing a self-limiting disease that includes a lot of vomiting.
12. This is an example of some of the skin and soft-tissue diseases that Staph produces. Impetigo is a disease that is commonly seen in young children; it can be caused by Staph, as well as Streptococcus pyogenes. This is typical of what you see here. You see a reddened area of the cell & a lot of “honey-crusted” scabbed-over skin lesions. Kids especially get it on their arms and legs. So the pathogenesis of this is that children, especially in the summer months, may be playing outdoors & wearing shorts. If a mosquito or another insect bites the leg or the arm, it itches. So the child scratches it. And if there is dirt on the hands or the fingernails, by scraping the skin you finish what the insect bite has already started, and you break the skin. Then these Staph that are present in the environment get into the intact skin, and they start causing these lesions.
Staph uses all of these different virulence factors that it produces to spread throughout the skin, and you get these coalescing lesions like you see here. Where you have a pus-filled lesion is a furuncle or a boil. If you get several of these spreading together like this, you can refer to them as carbuncles. So you can get little local abscesses or you can get conditions that we refer to as cellulitis, which is actually a spreading of inflammation without an obvious pocket of pus. This is a little furuncle on the eyelid, and all of this area on the face where you see this red, erythematous area is all cellulitis. And one of the bad things that you can get from something like this or from a direct invasion of the body is entrance in the bloodstream where the bacteria can travel throughout the body. And when they do that, you run the risk of getting the organisms settling on a heart valve and causing endocarditis. And that’s a very serious consequence. Anytime you have a damaged heart valve (or even if you don’t have a damaged heart valve), if you get a virulent organism in the bloodstream, there’s always the concern that the bacteria can land on the heart valve and multiply, ultimately damaging the heart valve and causing heart failure. Occasionally you will see Staph as the cause of brain abscesses or meningitis or even epidural abscesses.
We had a case that I heard about just last month from an elderly man who was having a lot of back pain due to osteoarthritis. So one of the treatments they do for back pain is they give an epidural block. They will inject steroids and anesthetics into the spinal area, into the epidural space to relieve the pain. Well, this man had this block, and shortly after he developed an abscess at the site of the epidural injection. And he had a severe and protracted hospital course as a result of this. Ultimately they showed the abscess by doing x-rays of the spine, and you could see a large developing cavity of abscess. This had to be drained surgically, and it was S. aureus that had almost certainly been introduced into this area when he had the injection. So it was a complication of the injection that he had that he got this epidural abscess that he had.