LESSON 2.1 WORKBOOK What does it mean to have an infectious

Transcription

1 Salmonella Typhimurium LESSON 2.1 WORKBOOK What does it mean to have an infectious disease? In Unit 1, we learned that infectious agents like bacteria, viruses and parasites cause infectious diseases. Now we are going to focus on how infectious diseases are identified. We will explore the distinction between correlation and causation, and how it is sometimes very difficult to establish causation. Determining that a disease is caused by an infection is also surprisingly difficult. Although identifying the pathogen is crucial for both treatment and prevention it is a relatively new skill, largely because identification usually requires specialized tools such as microscopes. How do you know if a disease is infectious? Inectious diseases can be caused by all manner of microbes, including viruses, bacteria, fungi, protozoa, and multicellular parasites. As diverse as these organisms are, it is unsurprising that they have equally diverse ways of infecting us in the first place. Some microbes are only infectious through the exchange of blood or other bodily fluids (HIV), while others can be transmitted by skin to skin contact (syphilis, HPV), and still others can be inhaled from tiny droplets in the air (tuberculosis, influenza). Exposure to a microbe means something very different from one microbe to another. For example, being in close contact with a cholera victim may not actually expose you to the cholera bacterium, whereas swallowing water contaminated by the feces of a cholera victim would. Conversely, being in close contact with a tuberculosis victim could definitely expose you to the tuberculosis bacterium. This certainly complicates things for researchers and doctors, who have to determine who has or has not been exposed and to what! Lesson 2.1 1

2 DEFINITIONS OF TERMS Correlation: a connection between two things. Causation: When a change in one thing results in a change in another. For a complete list of defined terms, see the Glossary. LESSON MATERIALS How do we know whether a disease is infectious? As we try to uncover how a disease is transmitted, another complication is that during our daily lives we are exposed to many different microbes. Even the simple act of taking the subway may expose us to microbes coughed into the air and to bacteria rubbed onto handles from poorly washed hands. With so many potential exposures, how do we determine which one caused an infection? This difficulty introduces the concept of correlation versus causation. To illustrate this point, imagine an epidemic that affects all the occupants of a hotel. Let s imagine that all the sick people in the hotel watched the same TV program. Watching the program is correlated with getting the infection. But it does not mean that the TV program caused the infection. Although in this case it is obvious because a TV program could not cause a disease, it might not have been so intuitively obvious if we had used the example of all the occupants eating the same breakfast. We will return to the important concepts of correlation and causation throughout this unit. What do we need to do in order to identify a pathogen? It may not be easy to find a pathogen or the source of a disease. In fact, identifying the source of an infection is often quite difficult. For example, during the years of the black plague, people knew that where there were many cats there was plague. Many people mistakenly believed that this correlation meant that the cats were causing the disease, when in fact the cats were hunting the rats that carried the fleas that were the true carriers of the plague bacteria. By destroying the cats, they inadvertently made the plague worse! Identifying the source of a pathogen is particularly difficult when we have no way of actually seeing the pathogen. This was an enormous problem before the invention of the microscope. Imagine telling someone from before the age of microscopes that disease was caused by something odorless, tasteless and invisible. Would they believe you? In addition, where there is one kind of microbe, there often thousands more different types. For example, fifty percent of the dry weight of human feces are bacteria. Imagine trying to identify which type of bacteria is causing gastrointestinal disease! Is finding a microbe proof that it causes the disease? If you can t find a microbe is that proof that the disease is not infectious? Figure 2.1.1: Spores lie dormant and then become activated once they Is cancer caused by an infectious agent? Lesson 2.1 infect a host. 2

3 DEFINITIONS OF TERMS Gastroenteritis: Inflammation and pain of the stomach and intestines. For a complete list of defined terms, see the Glossary. LESSON MATERIALS Typhoid illustrates the difficulties of identifying the source of an infectious disease. Typhoid is a waterborne disease caused by the bacterium Salmonella typhi. Unlike Vibrio cholera, which reproduces in the intestine (remember this is technically outside the body) S. typhi enters the inside of body, and then replicates inside cells. Annually, million people are infected with typhoid, and of those, at least a quarter of a million will die. Typhoid mainly targets children and young adults between 5 and 19 years old, and infection occurs by eating food or water that is contaminated with feces from an infected person. The main symptoms of typhoid include a slowly progressing fever that can reach 40 C (104 F), profuse sweating, gastroenteritis, and green diarrhea with a pea soup smell. Less commonly, victims may develop a rash of flat, rose-colored spots, and in severe cases, internal bleeding can occur and prove fatal. This carries on into the fourth and final week of infection when, if the patient survives, the fever symptoms begin to resolve. While V. cholera can easily be isolated for identification merely by putting a stool sample under the microscope, isolating S. typhi is much more complicated for a number of resons. First, unlike cholera, only a very small number of bacteria cause the infection and they are often found actually inside host cells. This means that bacteria have to be identified from samples from the blood, bone marrow or stool of the infected person by placing the samples in a nutrient broth that the bacteria can feed off. Alternatively, antibodies against salmonella flagella may be used to identify the bacteria, but this is only an option if the patient has been ill for at least two weeks because it takes that long for antibodies to develop. Meanwhile the patient may be entering the critical third week of infection when fatal complications can occur. Because of this, developed countries often start treatment with antibiotics even before S. typhi is positively identified. Typhoid Mary was not sick but people she cooked for got typhoid. An asymptomatic carrier suffers no symptoms from disease, but is capable of infecting others. The most famous asymptomatic carrier was Mary Mallon, commonly known as Typhoid Mary, a young Irish cook at the turn of the century who was responsible for infecting at least 53 people with typhoid, three of whom died from the disease. It was difficult to prove that she had infected people with typhoid since she herself was healthy. In fact, when confronted, she denied both that she was infected with typhoid and that she played a role in spreading the disease - and refused to stop working as a cook! As a consequence she was forcibly quarantined and even died in quarantine. Actually it is possible that she was born with the disease, as her mother had typhoid fever during her pregnancy. This would account for why she always vehemently denied Lesson 2.1 having had typhoid herself. Can you imagine being told that you were making people sick when you feel per- 3 fectly healthy? Describe three symptoms of Typhoid What are the important differences between typhoid and cholera? Why was it so hard to identify Mary as the source of the disease? Was isolating Mary justifiable?

4 STUDENT RESPONSES Remember to identify your sources. Ghost Map reading - How did they identify the source of the disease? Lesson 2.1 4

5 LESSON 2.2 WORKBOOK How can we prove a disease is infectious? Identifying bacteria and viruses As we discussed in lesson 2.1, being able to actually identify infection-causing microbes is a relatively new skill. One of the biggest reasons for this is that identification usually requires special tools such as microscopes. With the invention of the microscope, our understanding of infectious disease increased dramatically. Today, what we know about microbes continues to change as technology and science develop new and exciting tools to investigate microbes. Correlation is not always enough: we need to see the aggressor! Since you cannot see microbes by eye, how would you know that life can be microscopic? Before the invention of microscopes that were strong enough to visualize microbes, the idea of microscopic organisms was simply preposterous. Figure 2.1.1: Van Leeuwenhoek s flea - the first microbe seen under a microscpoe. Lesson 2.2 1

6 DEFINITIONS OF TERMS Correlation: a connection between two things. Causation: When a change in one thing results in a change in another. For a complete list of defined terms, see the Glossary. LESSON MATERIALS Indirect evidence that small life forms exist: debunking spontaneous generation. Until the 1700s it was commonly believed that living organisms such as flies or bacteria could be generated from non-living matter like decaying meat. This was callled the theory of spontaneous generation. Then, scientists started to question those beliefs and in 1668 the Italian physician Francesco Redi performed experiments to test whether it was really true. Redi wondered whether the flies actually arose from eggs laid by other flies that had landed on the meat. To test his hypothesis, he performed the experiment shown below. First he placed rotting meat in a series of jars, and then he covered some of them, thus preventing flies from landing and laying their eggs. As he expected, the meat in the uncovered jars was soon teeming with maggots, whereas the meat in the covered jars was maggot free. Figure 2.2.2: The experiment that proved life can t be generated spontaneously. Could Franceco Redi s experiment be used to prove the theory of spontaneous generation if milk spoilage was studied? Lesson 2.2 2

7 LESSON MATERIALS The development of the microscope: The story of Antonie van Leeuwenhoek Although lenses have been used for magnification since ancient times, it was not until relatively recently that the microscope was perfected to an extent that we could view microbes and especially bacteria. The early lenses could not achieve a magnification of more than ten fold. Recall from Unit 1 the relative size of a bacterium to say, the width of a human hair. The average human hair is 100 µm (microns) wide, while the average bacterium is 1-10 µm (microns) in length. So, a magnification of 10X would not be sufficient to make most bacteria visible to the human eye. This all changed in the late 1600s. Antonie Van Leeuwenhoek, a Dutch tradesman and scientist who is often referred to as the father of microbiology for his discoveries with the microscope, developed a method of creating a lens that could magnify objects more than 200 times. His endless curiosity lead him to examine everything in his world with his microscopes, including plants, insects, rainwater, and even his own dental plaque! He was the first person to actually see and describe bacteria, although this discovery did not translate into germ theory (this is discussed below) for many years. Fig 2.2.3: Van Leeuwenhoek was the first to invent a microscope that could detect microbes. How much smaller than a flea is a an average bacterium if we estimate that the flea is one millimeter in diameter? Lesson 2.2 3

8 LESSON MATERIALS The microscope showed people a world of microbes If you take a sample and look under the microscope you see a zoo of microbes. After the development of the high power microscope, it was possible to see many bacteria. However, it was not instantly clear that these animalcules could cause disease because microbes were seen to be everywhere. Bacteria are astonishingly diverse - in your mouth alone, there can be as many as 700 different species of bacteria! To add another challenge, each person s mouth has DIFFERENT species of bacteria. If only two or three of those species are pathogenic, how could you possibly isolate them from the other 698? Early microbiologists clearly needed to be able to isolate single species of bacteria. In the 1800s Louis Pasteur and Robert Koch, two of the founders of modern microbiology, experimented with many methods of growing bacteria, including broths made from urine or meat extracts, and potato slices. Fannie Eilshemius, the wife of Robert Koch s assistant, suggested mixing the nutrients into an agar gel, and letting the bacteria grow on top, a method still used for bacterial culturing today. Altering the nutrients in the broth changes which baceria will grow. The final proof that spontaneous generation does not exist. The realization that microbes exist allowed scientists to debunk the spontaneous generation theory once and for all. In the late 1700s, the Italian scientist Lazzaro Spallanzani placed nutrient broth in two separate bottles and covered only one. Days later, the uncovered bottle was teeming with life, whereas the covered bottle remained microbe-free. However proponents of the spontaneous generation theory argued that spontaneous generation hadn t worked in the covered bottle simply because it was deprived of air. In the mid 1800s Louis Pasteur, a French chemist and microbiologist, provided final proof. He boiled meat broth in flasks to kill off any microbes. Then he left some spouts to the flasks open to both air and dust Other flasks had long swan-like necks that allowed air, but not dust or other particles to enter. Soon the broth in the open flasks was filled with microbes, but the broth in the swan-necked flasks remained clear. The experiment showed not only that microbes come from other microorganisms, but also that microbes are everywhere, even in the dust in the How could growing microbes help to identify a pathogen? Define germ theory air! Pasteur s experiments also provided evidence that doctors should sanitize their hands and equipment when Lesson 2.2 treating patients and helped to lay the groundwork for modern germ theory. 4

9 LESSON MATERIALS With their new knowledge about microbes and the ability to isolate bacteria by growing them on nutrient agar plates, people can grow millions of bacteria from one single parent bacterium. This allows scientists to isolate a single type of bacterium from the mixtures found in nature. The Chamberland filter and the discovery of the virus After Pasteur, scientists continued to investigate other properties of microbes. Charles Chamberland, a French microbiologist who worked with Louis Pasteur, found that in some cases nutrient broth remained infectious even after he had filtered it to remove bacteria and dust particles. This was a puzzle - where was the infection coming from? Recall from the previous unit that viruses are, on average, one one-hundredth the size of bacteria. They are too small to see, even with the strongest of conventional microscope lenses. So this new type of infection couldn t be seen. What was it? In 1892, a Russian scientist named Dimitri Ivanovski was trying to isolate an infection that was decimating tobacco crops. Following the wisdom of the day, he assumed that the culprit was a bacterium, and so he tried to isolate it by filtering a broth made from the sap of the infected plants with the Chamberland filter. Imagine his surprise when bacteria separated from the sap by the filter did not cause disease, while the purified sap did! He came to the conclusion that the disease was being caused by an infectious agent much smaller than bacteria. Then, in 1898, A Dutch scientist, Martinus Beijerinck, independently discovered that a bacteria-free medium could still contain an infectious agent. He coined a term to describe this tiny microbe: virus. How did Dimitri Ivanovski show that an infectious agent can be smaller than a bacteria? Would you expect the viruses isolated from the tobacco plants to actually grow in the broth like the bacteria? Why or why not? Lesson 2.2 5

10 STUDENT RESPONSES Calculate the size of most viruses relative to bacteria, given the following information: Remember to identify your sources. A bacterium is approximately 1 micron in diameter and a virus is 100 times smaller than a bacterium. 1 micron = 1 X 10-6 meters, or meters. 1 nanometer = 1 X 10-9 meters, or meters. Lesson 2.2 6

11 Bacillus anthracis LESSON 2.3 WORKBOOK Lesson 2.3 Identifying infectious bacteria: Correlation and causation In the last lesson we learned how the invention of microscopes allowed bacteria to be observed for the first time, allowing researchers to discover that disease can be transmitted by creatures too small to see. We also learned how bacteria can be cultured and how the invention of superfine filters allowed viruses to be identified. But how do we actually prove that a particular microbe is the cause of a specific disease? Here we will draw on what we have learned in previous lessons to establish a set of principles to test whether a microbe causes a particular disease. If a disease is infectious what will you see? As we have discussed before, people have known for a long time that some diseases can be transmitted from one person to another, they just didn t know how. Here is the ancient Greek historian Thucydides, describing the infectiousness of the plague of Athens in the 5th Century BC: People in good health were all of a sudden attacked by violent heats in the head, and redness and inflammation in the eyes, the inward parts, such as the throat or tongue, becoming bloody and emitting an unnatural and fetid breath. These symptoms were followed by sneezing and hoarseness, after which the pain soon reached the chest, and produced a hard cough. When it fixed in the stomach, it upset it; and discharges of bile of every kind named by physicians ensued, accompanied by very great distress. In most cases also an ineffectual retching followed, producing violent spasms, which in some cases ceased soon after, in Figure 2.3.1: A mass grave from the plague of Athens. others much later. (over...) Lesson 2.3 1

12 DEFINITIONS OF TERMS Correlation: a connection between two things. Causation: When a change in one thing results in a change in another. For a complete list of defined terms, see the Glossary. LESSON MATERIALS Externally the body was not very hot to the touch, nor pale in its appearance, but reddish, livid, and breaking out into small pustules and ulcers. But internally it burned so that the patient could not bear to have on him clothing or linen even of the very lightest description; or indeed to be otherwise than stark naked. What they would have liked best would have been to throw themselves into cold water; as indeed was done by some of the neglected sick, who plunged into the rain-tanks in their agonies of unquenchable thirst; though it made no difference whether they drank little or much. Besides this, the miserable feeling of not being able to rest or sleep never ceased to torment them. The body meanwhile did not waste away so long as the distemper was at its height, but held out to a marvel against its ravages; so that when they succumbed, as in most cases, on the seventh or eighth day to the internal inflammation, they had still some strength in them. But if they passed this stage, and the disease descended further into the bowels, inducing a violent ulceration there accompanied by severe diarrhea, this brought on a weakness which was generally fatal. For the disorder first settled in the head, ran its course from thence through the whole of the body, and, even where it did not prove mortal, it still left its mark on the extremities; for it settled in the privy parts, the fingers and the toes, and many escaped with the loss of these, some too with that of their eyes. Others again were seized with an entire loss of memory on their first recovery, and did not know either themselves or their friends......by far the most terrible feature in the malady was the dejection which ensued when any one felt himself sickening, for the despair into which they instantly fell took away their power of resistance, and left them a much easier prey to the disorder; besides which, there was the awful spectacle of men dying like sheep, through having caught the infection in nursing each other. This caused the greatest mortality. On the one hand, if they were afraid to visit each other, they perished from neglect; indeed many houses were emptied of their inmates for want of a nurse: on the other, if they ventured to do so, death was the consequence. ( In this case, Thucydides described the disease as being infectious because those who visited and cared for the sick fell ill themselves. As we saw in the Ghost Map reading, the link between exposure and sickness is not always so clear. Not all communicable diseases have such a distinct mode of transmission. If someone coughs on you and you get sick, it is intuitively easy to say this is what made me sick. But can you be sure? Figure 2.3.2: Thucydides If an infectious disease is transmitted from human to human, what would you expect to observe over time and why? What clues does Thucydides account give you about possible routes of transmission? Lesson 2.3 2

13 LESSON MATERIALS Think about what we have seen so far in the course. What do infectious diseases have in common? How would you know that a given disease is infectious? Consider athlete s foot. Athlete s foot is a transmissible infection caused by a parasitic fungus that lives in the skin. It is transmitted when an infected person contaminates surfaces that others touch with their bare skin, like the floor in a public shower. Would you say that it is an infectious disease because it can spread? How does this differ from poison ivy? The poison ivy rash is caused by exposure to a toxin produced by the plant Toxicodendron radicans. Both are caused by skin coming into contact with something. So what makes one an infectious disease but not the other, and how can you tell the difference? We need to prove that the disease can spread. The reason that athlete s foot is an infectious disease whereas poison ivy is not is that poison ivy is caused by a toxin from a plant, rather than a replicating microbe. More importantly, poison ivy is not contagious; you cannot give poison ivy to another animal, you must directly contact the oil from the plant. Conversely, athlete s foot can spread from person to person quite easily. Just think, if one person on a basketball team gets athlete s foot, the others will soon follow! We need to be able to associate the disease with a source of infection. This requirement is relatively straightforward; all people with the disease need to be exposed to the same source of infection. If you theorize that contaminated water causes an illness, then sick people must be exposed to the same contaminated water. Inversely, if a group of people becomes ill and they all use the same water, then it is a possible source of contamination (remember the Ghost Map). However, just correlating the symptoms with the exposure does not prove that the disease is caused by the exposure. For example, people who use common water may have other things in common such as listening to the same music. Just because the shared water is more plausible as a cause of disease than music doesn t make it true. We need to isolate an infectious agent from all infected individuals. If some people with a disease are not infected with the expected microbe, can the disease be caused by that microbe? Say you are trying to prove that Lyme disease is caused by a bacterium passed to humans through ticks. You must be able to find that bacterium both in the tick and Figure 2.3.3: Top: Athletes foot. Bottom: Lesson 2.3 in the infected human. But some bacteria are hard to grow Poison ivy 3 - they might be present but you just can t see them. Can you think of any eamples where an infectious disease is not transmitted from human to human? If we find that people with a disease are infected with a certain microbe would we be justified in saying that the microbe caused the disease? Why or why not?

14 LESSON MATERIALS We need to show that the infectious agent that we isolated can cause the disease. You will need to isolate the microbe and then use it to infect the person, animal or other host you are proposing will get the disease, and then show that the infection produces the symptoms of the disease in the new host. This requirement is often the most difficult to fulfil - its obviously unethical to deliberately infect someone. To get around this scientists often use animals as models of disease. But sometimes an animal model is hard to find - armadillos are the only model of leprosy for example. Correlation versus Causation Perhaps the most common mistake when interpreting data is to assume that correlation means causation. Although there will always be a correlation between a causative agent and its outcome, most correlations are not causative. For example say there is an epidemic in a neighborhood that shares a common water supply, but the neighbors also all drink milk supplied by the same company. Both the water supply and the milk supply are correlated with the disease, so how do we decide which one is causing the illness? Isolating a mcirobe from sick people that is also found in the milk or the water is a start, but still not proof - people, milk and water probalby share millions of microbes - we can only prove causation by using a single microbe to cause disease in healthy hosts. The tragedy of mistaking correlation for causation: You can see how tough it is to prove that a particular exposure causes disease! This has resulted in grave errors in both directions. For example: It is now widely accepted that tobacco can cause lung cancer, but for a long time the tobacco industry used scientific studies as proof that it didn t. They are able to do this legitimately because we can only make definite statements about how much tobacco alters the probability of getting cancer. For the latter half of the twentieth century, tobacco companies capitalized on this technicality and claimed that the inability to absolutely prove a causal link means that tobacco is harmless. As evidence they would say that non-smokers get lung cancer and many smokers never get lung cancer, therefore. Figure 2.3.4: The bad science that linked vaccines with autism has caused a public health nightmare. More recently another mistake of correlation for causation has had horrible consequences. Both the use of vaccines and the diagnosis of autism have increased dramatically since the 1930s, and many concerned parents have mistaken this correlation for causation based on poor evidence that was highly publicized. The resulting mania has led to parents refusing to provide their children with the MMR vaccine (Measles, Mumps and Rubella), which prevents serious and highly infectious diseases that often result in death or permanent injury. The dangerous consequences of the misinterpretation do not only affect the people who actually made the mistake. Lesson 2.3 The thousands of unvaccinated children put those they are in contact with at risk as well as themselves. In 4 addition, this controversy has diverted valuable resources away from other promising areas in autism research. How would you show that a microbe actually caused a disease (causation?) If vaccines did cause autism what kind of predictions could you make about vaccination?

15 DEFINITIONS OF TERMS For a complete list of defined terms, see the Glossary.. LESSON MATERIALS The first person to formalize a process to demonstrate that a disease is caused by an infectious agent was Robert Koch. Robert Koch was a German physician at the turn of the 20th century whose research won him the Nobel Prize in He successfully isolated the anthrax, tuberculosis, and cholera bacteria and proved that anthrax spores in the soil are the source of a severe wasting disease in cows. He is known as one of the fathers of microbiology. His other major contribution was to develop a set of four criteria he called postulates that have to be satisfied in order to definitively prove a causative relationship between a microbe and a disease. Koch s postulates are summarized below: Figure 2.3.5: Robert Koch was the first to describe the principles behind infectious disease. Robert Koch s postulates does the microbe cause disease? 1. Associa>on It must always be present in every case but not in healthy animals. 2. Isola>on It must be isolated from the sick animal into into pure broth (culture). 3. Causa>on The pure microbe must cause the disease in a healthy animal. 4. Re- isola>on - When the microbe is re- isolated from the sick animal it must be the same as the original. What is an example of a disease we have seen earlier that does not exactly fit Koch s postulates? Lesson 2.3 5

16 STUDENT RESPONSES Remember to identify your sources. In you own words describe each of Koch s postulates: Lesson _

17 Bacillus anthracis spores LESSON 2.4 WORKBOOK Identifying infectious bacteria: Koch s postulates and cholera This lesson grapples with the problem of arriving at causation from correlation. In this lesson, we will revisit Koch s postulates and learn how he used them to prove that Anthrax bacillus caused disease. You will also have a chance to apply Koch s postulates to another disease that we have already covered, Cholera. Throughout this lesson, consider potential challenges to fulfilling these postulates. Correlation and causation: How can we determine whether a microbe causes a disease or whether it is simply associated with a disease? In the last few lessons we have discussed patterns of infection. As we have seen, some diseases are passed directly from person to person through contact with bodily fluids, air, or skin. Other infections have an environmental or animal intermediate, for example cholera is usually transmitted when excrement of an infected person contaminates drinking water. Still other diseases, such as malaria, yellow fever, or Lyme disease, pass from person to person through a non-human vector such as a mosquito or a tick. Koch s postulates provide us with a valuable tool for identifying the causes of infectious disease. When the cause is identified the disease my be prevented or better treated. Here we will focus on the processes that Koch used to show that Anthrax is caused by the bacterium Bacillus Anthracis and that Cholera is caused by Vibrio cholera. Review the difference between correlation and causation. Review Koch s postulates. Lesson 2.4 1

18 DEFINITIONS OF TERMS Correlation- a connection between two things. Causation- when a change in one thing results in a change in another. For a complete list of defined terms, see the Glossary. LESSON MATERIALS Isolating Anthrax: the development of Koch s postulates Robert Koch was a Prussian physician who graduated from medical school in In the region where he worked, farmers had a serious problem with severe epidemics in cattle. The symptoms were severe and ultimately fatal, beginning with staggering and trembling, and progressing to convulsions, bleeding from body openings, and ultimately a quick death. It was a puzzle because the disease didn t obviously spread from one cow to another. We now know that the Anthrax bacteria causing the disease (Bacillus anthracis) survive in hostile environments by making spores. The spores remain on the grass where they are eaten by cows. Once the cows eat the spores, the bacteria recover from dormancy and begin to replicate quickly, making the cattle sick. At the time of Koch s work the anthrax bacillus bacteria had been identified, but had not yet been established as the CAUSE of the disease. #1. Association: It must always be present in every case- but not in healthy animals When Koch took blood samples from cattle infected with anthrax, they always had large numbers of Bacillus anthracis. But the cattle didn t infect each other, so the question was; how was the Bacillus anthracis involved? Was it an important causative agent, or was it just there by coincidence? #2. Isolation: It must be isolated from the sick animal into pure culture Koch filtered the bacteria out of the blood of infected cattle, grew them in a broth and isolated the anthrax bacteria from the mixture. Remember, there are ten bacteria in the body for every host cell, so simply finding the bacteria in the blood alone does not prove causation. #3. Causation - the pure microbe must cause the disease in a healthy animal Koch then injected the anthrax-containing broth into a healthy animal. This cow got sick with the same symptoms as seen in the epidemic! This step is why isolation of a pure culture of bacteria is required. If he had used a mixture of bacteria he would not have been able to determine which one caused the disease. #4. Re-isolation- when the microbe is re-isolated from the sick animal it must be the same as the original. Koch filtered the bacteria out of the second set of sick cows, and then isolated Bacillus anthracis again. This step provided another layer of control. For example re-isolation helps to rule out the original culture being contaminated with something else because you d be unlikely to isolate the same bacteria twice. Koch proved his point by using the same principles to isolate other infectious microbes such as the bacterium that causes tuberculosis, Mycobacterium tuberculosis, in 1877, and the bacterium that causes cholera, Vibrio cholera in For Figure 2.4.1: Koch proved that bacillus anthracis was the cause of the cow-wasting disease. Can you think of exceptions to Koch s first postulate that we have seen in this course? this work he was awarded the Nobel Prize in Lesson 2.4 2

19 ! Vibrio cholera DEFINITIONS OF TERMS Phytoplankton: microscopic plants and algae that live in water. For a complete list of defined terms, see the Glossary. LESSON MATERIALS Applying Koch s postulates to cholera. Review of cholera: Vibrio cholera normally inhabits tropical coastal estuaries such is in Bangladesh, where it lives in close association with phytoplankton. Humans can be infected when they enter this ecosystem and eat or drink contaminated food or water. High temperatures can lead to blooms when Vibrio replicates rapidly, increasing the likelihood of transmission and epidemic spread. In fact, we can think of the diarrhea that V. cholera causes as an evolutionary mechanism that helps cholera bacteria spread to new hosts. Association: The association postulate seems pretty straightforward; is the microbe present when there is disease? In the top picture, we see a man sick with cholera. The bucket beneath his bed is filled with rice-water stool which is a term used to describe the diarrhea of cholera victims. The rice-water stool is so-called because it is filled with white flakes resembling rice that are actually sloughed off pieces of the epithelium of the small intestine. Also inside that bucket are billions of V. cholera bacteria. The limitations!" #$$%&'()%*!"!#$!%&'$!()*(+'!,-!./-'-0$!10!-2-/+!3('-!"!,&$!04$!10!5-()$5+!(01%()'6!! +",$%-()%*!"!#$!%&'$!,-!1'4)($-7!8/4%!$5-!'139!(01%()!10$4!10$4!.&/-!3&)$&/-6!." /(0$()%*!"!!:5-!.&/-!%13/4,-!%&'$!3(&'-!$5-!71'-('-!10!(!5-()$5+!(01%()6!! 1" 234'$%-()%*5!;!<5-0!$5-!%13/4,-!1'!/-;1'4)($-7!8/4%!$5-!'139!(01%()!1$!%&'$!,-!$5-!'(%-!('!$5-!4/1=10()6! A scientist looking for a microbial culprit for cholera would find abundant bacteria in the rice water stool. One problem is that V. cholera isn t the ONLY bacteria that will be found in the bucket. Can you think of another potential problem in establishing association? Do you think that Vibrio would be found in the stool of healthy individuals? Isolation: In order to implicate a microbe, we need to isolate it from the commensal bacteria with which it will be found and grow it in pure culture. Figure 2.4.3: Vibrio cholera is easily isolated from The picture to the left shows the isolation of chol- Lesson 2.4 the stool. 3 era from rice water stool. >(39!!"#$%6?!@AAB! Figure 2.4.2: The intestinal epithelia found in the stool of cholera patients makes it look like rice. What are some potential problems that might arise when looking for an association? What are some potential problems that might arise when isolating bacteria from an infected individual?

20 ! For a complete list of defined terms, see the Glossary. LESSON MATERIALS The limitations Not only do you have to separate your candidate microbe from the zoo of microbes co-exisitng within the patient, you must also create an environment such as nutrient broth, agar gels, etc. that will support the growth of that microbe. This can be very tricky - surprisingly enough even now scientists only know how to grow about 30% of all bacteria! Important bacteria we still can t grow in culture include Yersinia pestis that produces plague and Borrelia burgdorferi that produces Lyme disease. Causation: To prove causation, the microbe you isolated must also cause disease in a healthy subject. This can prove problematic with microbes that only infect humans (you won t have an animal model and you can t intentionally infect humans with cholera!). It is also problematic in that some people are more susceptible to certain diseases disease than others. Remember Typhoid Mary? Typhoid is an example of a bacterium that doesn t always cause symptoms. Differing susceptibilities may be due to immune system status, age or general health. Figure 2.4.4: The microbe must cause disease in a healthy animal. Re-Isolation: Let s assume you have a convenient animal model for cholera like a mouse. By infecting the mouse and then re-isolating the microbe from it when it gets sick, you are attempting to confirm that the microbe that you isolated did in fact cause the disease in your test animal. If you infect a mouse with a bacterium that you isolated and they do get sick, but then their diarrhea does not contain the vibrio cholera that is your original candidate, it is possible that your initial identification of the candidate was mistaken or that your isolation What might prevent you from showing causation? What does it mean for Koch s postulate of causation if only some people become sick after an infection? Figure 2.4.5: Reisolating the same bacteria is further proof that it is the cause of disease. became contaminated. Lesson 2.4 4

21 Helicobacter pylorii DEFINITIONS OF TERMS Peritonitis: inflammation of the peritoneum, the thin tissue that lines the inner wall of the abdomen and covers most of the abdominal organs. For a complete list of defined terms, see the Glossary. LESSON 2.5 WORKBOOK Identifying infectious bacteria-ulcers, and what s up your nose? This lesson continues to grapple with the problem of arriving at causation from correlation by looking at another infectious agent that does not perfectly fit into Koch s postulates, helicobacter pylori that is thought to cause stomach ulcers. Despite the Nobel Prize having been given to its discoverer, Koch s postulates have never been fully established for this infectious agent. Hence we can only say that helicobacter is the expected cause of ulcers. Pathophysiology of a stomach ulcer. An ulcer is a persistent wound in the epithelium of the stomach or small intestine that does not heal. Stomach (peptic) ulcers are particularly painful because of the strong acid secreted into the stomach during a meal. The stomach epithelia are normally resistant to the acid, but when they are damaged they expose parts that are acid sensitive. As a result, peptic ulcers cause the most pain during a meal. Duodenal ulcers Duodenal ulcers are found in the small intestine below the stomach. In this case eating food soothes the pain because the digested food protect the exposed damaged epithelia. In both cases, symptoms may include vomiting and in extreme cases peritonitis, which results from bacteria entering the sterile area of the abdomen through the ulcer. Up until 1982, Ulcers were thought to be caused by Fig 2.5.1: Peptic ulcers are found in the stomach, duodenal ulcers are found in the small intestine. What accounts for the differences in when you feel pain if you have an ulcer? How might an ulcer lead to peritonitis? Lesson 2.5 genetics, stress and/or diet. 1

22 DEFINITIONS OF TERMS Correlation: a connection between two things. Causation: When a change in one thing results in a change in another. For a complete list of defined terms, see the Glossary. LESSON MATERIALS Case Study: Stomach ulcers and the mysterious bug Background: Generally healthy, although a little overweight, Reg began to have problems with his digestion after work at the supermarket chain became stressful: There was some trouble over a missing piece of jewelry and the relationships between the staff became very strained. As the General Manager of the large store where it happened, I took the brunt of everyone s bad feelings. For the first time in my life, I experienced real stress. The woman whose expensive gold charm bracelet had gone missing accused a colleague of stealing. The bracelet never turned up, and both women left after a few weeks, but not before some intense bouts of recrimination and uncomfortable silences, all of which created a very bad atmosphere. Reg ignored the abdominal pain that came in the middle of the night, and began drinking a bit more than usual. Once things got back to normal at work after the two co-workers left, he expected to feel OK again, but the pain was worse than ever. I was not sleeping and I started to feel uncomfortable during the day as well. Reg said. His doctor suspected that he might have an ulcer and sent him for some tests. My brother and sister both have a history of ulcers, so I wasn t really surprised. I had never been troubled before but it puzzled me that the specialist at the hospital was so interested in my bad breath. I am famous for it and none of my grandchildren will come and sit on my knee for a cuddle, they say I am Granddad Firebreath, laughed Reg. Reg s bad breath had been with him as long as he could remember even though he had taken good care of his teeth, which were better than most men of his age. The test results revealed the Reg did indeed have an ulcer, and his blood showed high levels of antibodies to a bacterium Helicobacter pylori. Describe Reg s symptoms Lesson 2.5 2

23 STUDENT RESPONSES ID number Section Date _ How do you think the doctor confirmed that Reg had an ulcer? _ Stomach ulcers have been a health problem throughout history. There was some evidence from the case study that they may be an infectious disease. What is it? Would you be satisfied with this evidence as proof that stomach ulcers are an infectious diease? If you wanted to explore the possibility that stomach ulcers are caused by an infectious agent what steps would you take? Be as specific as you can. (Think about Koch s postulates: Association, Isolation, Causation, Re-isolation). _ Lesson 2.5 3

24 STUDENT RESPONSES ID number Section Date _ Where would you expect to find any microbes that could be culprits? How would you see them? _ How would you isolate the microbe? If you can t grow the microbe in culture what do you do next? _ Let s say you can culture the microbe, how would you know that the microbe causes the disease? Would you use a human to test this? What if the microbe does not cause disease in animal models? _ Lesson 2.5 4

25 STUDENT RESPONSES ID number Section Date _ Reg s Treatment: I had thought that stomach ulcers developed because of stress but I was told that over 80% are caused by a bacterial infection. I had never heard of Helicobacter pylori, but apparently people can be infected in childhood and never know it. We grew up in the South side of Chicago, it was tough and conditions were hardly luxurious. I ve probably had the infection since then, but it is only now that it has made its presence felt, explains Reg. Reg was given triple therapy with three antibiotic drugs known to clear H. pylori infections and was tested again six months later. Not only had the ulcer cleared up but, to the delight of all the family, my bad breath had gone too! says Reg. Tests showed that Reg was clear of the bacterial infection and that his ulcer had healed well. Reg s story included a stressful experience at work that he thought caused his ulcer. Do you think that his stress contributed to the ulcer? If bacteria caused his condition why would stress make it worse? Lesson 2.5 5

26 Staphylococcus aureus DEFINITIONS OF TERMS Morphology: The physical form or structure of a microbe. For a complete list of defined terms, see the Glossary.. 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

27 LESSON MATERIALS What bacterial characteristics might be useful when trying to identify a microbe? DEFINITIONS OF TERMS Correlation: a connection between two things. Causation: When a change in one thing results in a change in another. For a complete list of defined terms, see the Glossary. 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

28 LESSON MATERIALS 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? DEFINITIONS OF TERMS Phytoplankton: microscopic plants and algae that live in water. For a complete list of defined terms, see the Glossary. 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

29 LESSON MATERIALS 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

30 LESSON MATERIALS 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

31 MRSA DEFINITIONS OF TERMS For a complete list of defined terms, see the Glossary. LESSON 2.7 WORKBOOK Lesson 2.7: Antibiotic resistance When you get sick with a bacterial infection a doctor can prescribe you an antibiotic that will probably help you to get better. However, we are fast entering a world where many pathogens are resistant to many of the antibiotics in our arsenal. Here, we will address some social and medical problems associated with antibiotic resistance. We will also explore the dangers, causes, and potential solutions to this important problem. Why are we worried about antibiotic resistance? Treating bacterial infections with antibiotics is becoming less successful because many bacteria have adapted mechanisms that prevent their normal susceptibility to antibiotics. This poses a threat that is much more serious than you may be aware of. Antibiotics are used during surgery, pregnancy, trauma, age related immune failure, and much more! You may have read headlines in the news talking about MRSA outbreaks in hospitals. As we began to explore in the last lesson, MRSA is the name given to the Methicillin resistant form of the common bacterium Staphylococcus Aureus. More recent forms of MRSA are resistant to multiple antibiotics that have been commonly used to treat Staph infections. MRSA infections are a major concern in hospitals, where there are numerous people with compromised immune systems or who are recovering from surgery. What is antibiotic resistance? Lesson 2.7 1

32 LESSON MATERIALS DEFINITIONS OF TERMS Natural selection - The process of increasing the level of offspring with an advantageous trait. This is a fundamental part of evolution. For a complete list of defined terms, see the Glossary. Growing concerns: the return of Tuberculosis For most of the 20th century, Tuberculosis in the United States was declining. Much of this decrease was attributable to the effective use of antibiotics. However recent years have seen a global increase in the incidence of multiple drug resistant tuberculosis or MDR-TB, which is resistant to two or more of the major front-line drugs used to treat tuberculosis infections. MDR-TB poses a major health concern because we are quickly running out of new drugs to treat and cure tuberculosis. Even more worrying is the development of XDR TB or extensively drug-resistant tuberculosis, which is resistant not only to the front line drugs, but also to the second-line drugs. Patients infected with XDR TB have only a 30% chance of clearing the infection and are at serious risk for death. Fig 2.7.1: The incidence of XDR-TB in the US Misuse of antibiotics is a major factor in building resistance. In other classes we have learned about how microbes adapt their structures in response to challenges, for example the bacteria that changed their flagella proteins to evade detection by the immune system. Likewise microbes can adapt following the challenge of antibiotic treatment. These adaptations give rise to proportion of the population that are now resistant to treatment with the drug. Then, through the process of natural selection the resistant bacteria will out-compete the bacteria that are less fit in the presence of antibiotics. When we use use antibiotics, we kill the bacteria that are most susceptible to them, leaving room and resources for the ones that are more resistant. Hence every time we use antibiotics we are encouraging bacteria to adapt to resist them. As a result, it is very dangerous to use antibiotics in situations when they are unnecessary. Today we inject antibiotics into livestock to increase their growth rate, and we also use antibiotics in cosmetics. In addition, prescribing antibiotics before identifying the microbial origin of an infection increases the odds of giving antibiotics to people who are infected with viruses - a futile response that again encouragies antibiotic resistance. These practices are so prevalent that antibiotic resistance in bacteria is rising rapdily to the extent that we are running out of potential treatments for serious infections - like MRSA. Why are we concerned about a few cases of XDR TB? Do you think giving cattle antibiotics to increase the rate of their growth is a good or bad idea? Explain your reasoning.. Lesson 2.7 2

33 . LESSON MATERIALS Is it viral or bacterial? Many bacterial and viral infections have symptoms that are nearly identical. For example, it is very difficult if not impossible to distinguish between Norovirus and Salmonella when considering the symptoms alone: Both have symptoms of nausea, vomiting, diarrhea, abdominal pain, headache and lethargy. Norovirus accounts for about 50% of food poisoning cases in the United States. It is sometimes referred to as stomach flu and it can be passed from person to person, as well as through food or water that is contaminated by feces. Norovirus is seldom fatal. Salmonella on the other hand is a bacterium that can be contracted from contaminated food or water, or from exposure to reptiles. In rare cases it can result in severe dehydration that leads to death. What do you do when faced with a set of symptoms that could be there result of a bacterial infection OR a viral infection? How can you weigh the costs and benefits and benefits of treatment? Some infections can be easily diagnosed, such as strep throat, but others require timely and expensive procedures. For minor illnesses, the most common way is to wait and see. Most viral infections are cleared in a few weeks so if you are still sick after that time it may be justified to pull out the antibiotic. How do you know what antibiotic to use? If you isolate Staph. Aureus as the cause of an infection how would you determine which type of antibiotic to treat with? And what may happen if you don t have this information? Not all antibiotics work in exactly the same way, so it is sometimes difficult to know whether an infection is resistant to an antibiotic because it is caused by a resistant bacterium or whether it is due to a virus that wouldn t respond to antibiotic. The most rigorous way to decide which antibiotic to use is to isolate the bacteria and then test for antibiotic susceptibility, like we did in the lab. Unfortunately, this is not always an option because pathogens can be difficult to isolate, as we have discussed before. In those cases, doctors will often use trial and error: try one antibiotic then change to another if it doesn t work, or use a few at once. As we have seen this can be a high stakes gamble. A summary of ways that you can limit antibiotic resistance. Taking the full course of medication. Avoiding low doses for extended times. Using them only for serious infections. Label each of the discs A - L. What do the clear circles around some of the discs mean? What do you think the difference between the sharp edges and the fuzzy edges is? Which antibiotic would you choose to treat the infection with and why? If this is a plate of MRSA, which of the discs is a candidate to be methicillin? Fig 2.7.2: A plate of bacteria being tested for their susceptibility to antibiotics. The discs are soaked with different antibiot- Lesson 2.7 ics. 3