Staphylococcus aureus Morphology: The physical form or structure of a microbe.. LESSON 2.6 WORKBOOK Diagnosing infections, and, what s up your nose? Now we have discussed the different requirements that must be met in order to prove that a microbe can cause a disease we can start to think about how you find out which microbe is making you sick. This lesson covers different strategies that can be used in the clinical diagnosis of an infection. Different infections can cause similar symptoms but different microbes often require completely different treatments. For example, treating a viral infection with antibiotics will not kill the pathogen, nor will treating malaria with antibiotics or antiviral drugs. Hence we need to be able to identify what is causing an infection. How would you distinguish between two different bacteria that cause the same symptoms? You wake up one morning with a scratchy throat. Your muscles feel weak and you can feel a fever coming on... Most infectious diseases have some common symptoms including fever, weakness, coughing, and more. So how can you determine which infectious agent is causing your symptoms? With so many possibilities the process of elimination is more powerful than direct testing for the presence of a specific pathogen. Think back to Unit One. We learned that many bacteria have specific structures that help them cause disease or evade the immune system. Many of those structures make the bacteria look drastically different from each other under a microscope in addition to their general morphologies like rods, spheres, or spirals. How many bacterial structures that are important in disease can you remember from Unit 1? What do they do? Lesson 2.6 1
What bacterial characteristics might be useful when trying to identify a microbe? Correlation: a connection between two things. Causation: When a change in one thing results in a change in another. Bacteria have three major morphologies: Spheres: cocci Rods: bacilli Spirals: spirella Fig 2.6.1: The structures of bacteria important in disease. This provides our first step: take a sample from the affected tissue and look under a microscope! Fig 2.6.2: The three common bacterial shapes, cocci, rods and spirals. However, being able to identify the morphology of a bacterium raises yet another challenge, how could you tell the difference between two spherical bacteria for instance? Lesson 2.6 2
Gram staining Gram staining can be used when bacteria have the same shape to narrow down the microbe to one of two major classes: Let s say your your candidate pathogen is a gram negative rod. There are countless rod shaped bacteria, what other structures could you use to narrow your search? Phytoplankton: microscopic plants and algae that live in water. Gram positive bacteria will absorb the blue/ purple Gram stain in their cell walls. Gram negative bacteria won t absorb the blue/purple Gram stain in their cell walls. Remember that the Gram stain reacts with murein and Gram-negative bacteria have an extra membrane that shields the murein in the cell wall. This could be important for treatment - remember Gram negative bacteria are resistant to many antibiotics. Again we are using process of elimination to exclude a large number of potential bacteria with a single experiment. Even with this information, you will still need to collect more data to allow you to make an educated guess about the identity of the pathogen, so the process of elimination continues. Fig 2.6.3: Gram staining can differentiate between two major classes of bacteria. Gram posi/ve Streptococcus Gram nega/ve Escherichia Coli Fig 2.6.4: Gram staining can differentiate between two major classes of bacterial Can you remember why gram negative bacteria are resistant to more antibiotics? Lesson 2.6 3
Flagella can also be used to narrow your search. Flagella can provide a rather conspicuous clue to help you decide whether two similar-looking bacteria are the same. For example, here are images of Vibrio cholera (top) and Salmonella typhi (bottom). Both are gram negative rod shaped bacteria. Once you have a candidate, antibodies can be used for positive identification. When we are exposed to a pathogen, our immune system often creates antibodies as a specialized defense. The antibodies will attach tightly to a specific pathogen using a lock and key mechanism that inactivates it (we will examine how in detail later on). The presence of these antibodies provides us with an opportunity to positively identify a pathogen without ever isolating it. Detecting a pathogen with an antibody screen like this is a more indirect way of screening for an infection than isolating the bacteria, but in some cases it may be the only option. For example, the test for Lyme disease uses antibodies to detect the bacterium in a blood sample rather than isolating the bacteria from deep within the nervous system or joints of the patient. Fig 2.6.5: Chlamydia antibodies detected with a green stain in a sample of genital tissue. Fig 2.6.4: Top: Vibrio cholera. Bottom: Salmonella Typhimurium Although it is convenient, this approach has a major limitation - the antibodies persist even if the infection is cleared, so it is impossible to tell whether the infection is active or is over. A good example is the TB test. It measures the immune response to TB rather rather than the presence of the bacteria, so anyone who has been exposed to TB will have antibodies whether or not they have an active TB infection. Moreover, the TB vaccine itself causes antibodies to be made, so people who have been vaccinated will have the antibodies even if they have never had a TB infection. What do you notice about the two bacteria in the picture to the left that could allow you to distinguish them under a microscope? Does the presence of chlamydia antibodies prove that the person s active infection is caused by chlamydia? Why or why not? If not, how would you prove it? Lesson 2.6 4
Using the process of elimination to isolate a microbe: The nasal swab lab Let s say that you use a microscope and the Gram stain to find that spherical Gram-positive bacteria (cocci) are causing a particular disease (in this case a skin infection). How could you further narrow down whether it is regular Staphyloccocus Aureus, antibiotic resistant Staphyloccocus Aureus (MRSA) or even Staphyloccus Epididimis? The answer is critical because each of them will need a very different antibiotic treatment. The nasal swab activity we started in the last lesson is a perfect example of another tool we have in our toolkit - we can use different growth conditions to distinguish between different bacteria. In the experiment, the first type of agar plate you used was beige in color because it is made from a rich nutrient mixture called Luria broth (LB) that contains: Peptides and amino acids Vitamins (including B vitamins) Trace elements (e.g. nitrogen, sulfur, magnesium) Minerals like sodium chloride We use a rich plate first so we will be able to grow as many different types of bacteria as possible. The second type of agar plate is red in color because it contains extra mannitol (a sugar) and a higher concentration of salt in addition to the nutrients that are present in the LB plates. This high salt concentration encourages the growth of some bacteria like Staphylococci while inhibiting the growth of others, making these plates selective. Fig 2.6.6: The top plate (LB) is a rich nutrient mixture. The bottom plate (MSA) is specific for Staph. Aureus. The mannitol in the plates has its own purpose. It is present together wirh a color indicator called phenol red. If bacteria growing on the mannitol plate can ferment the mannitol, the chemical reaction causes the phenol red to a change color to yellow. This is diagnostic for Staphylococcus Aureus. Although several different species of Staphylococcus can grow in the nose, for instance Staphyloccus Epididimis, Staphyloccus Epiderimidis and Staphyloccus Aureus, only Staph. Aureus can ferment mannitol and turn the red indicator color yellow. So this reaction allows us to distinguish between Staph. Aureus and other Staph bacteria. Why is this important? Some forms of Staph. Aureus called MRSA have mutated to become extremely drug resistant. The presence of MRSA means that careful precautions must be taken to prevent contamination, because if infection occurs it is very hard to treat. About 20% of the population have MRSA in their nose as part of their commensal bacteria without apparently suffering any effects. However these people should be very careful with hygiene so The plates used in the nasal swab are restrictive: How? You culture a bacterium on the MSA plate that can ferment the mannitol, how can you determine whether it is Staph. Aureus or MRSA? Lesson 2.6 as not to infect others who may be more susceptible. 5