1. Outline of Lectures 18 Review of the work on contagious disease by Koch. 19 Epidemiology and Public Health. 20 Microbial interactions with higher animals. Examination of the normal flora of animals. 21 Entry of the pathogen into the host. Colonisation and growth. 22 Transmission of pathogens. Bacterial respiratory infections and sexually transmitted bacterial diseases. 23 Insect transmitted diseases. 24 Food-borne and water-borne bacterial diseases.

2. Course text: Brock Biology of Microorganisms Michael T. Madigan John M. Martinko Paul V. Dunlap David P. Clark Tenth/Eleventh/Twelfth (international) Edition Publisher: Pearson Education

3. 18. Review of the work on contagious disease by Robert Koch. Dr. Çiğdem Williams (chwill@essex.ac.uk)

4. Microorganisms as disease agents In the U.S., the major causes of death at the beginning of the 20 th century were infectious agents called pathogens. The elderly and the young were the most susceptible to the infectious diseases caused by these pathogens. Today, in developed countries the incidence of death is greatly reduced through understanding how these pathogens cause disease and the disease process itself, improved sanitary and public health practices and the development and use of antimicrobial agents. Instead non-microbial diseases (e.g. heart disease, cancer, etc.) are the major causes of death in the U.S. according to the data obtained in 2008.

5. Microorganisms and Disease in the USA Red: microbial disease Brock 12 th ed. Fig. 1.8 Green: non-microbial disease 1900 2008 Influenza and pneumonia Tuberculosis Gastroenteritis Heart disease Influenza and pneumonia Stroke Kidney disease Accidents Stroke Accidents Cancer Infant diseases Diphtheria Pulmonary disease Diabetes AIDS Suicide Cirrhosis of the liver Homicide Deaths per 100,000 population 0100200 100 200 0 Deaths per 100,000 population 280 205

6. Although, medical and microbial science have been successful in controlling many microorganisms and even eradicating them (smallpox), there are still microbial infections (malaria, tuberculosis, cholera, measles, pneumonia to count some) that cause death. Furthermore, diseases that could emerge suddenly [severe acute respiratory syndrome (SARS), bird flu, swine flu] and have the potential to infect different species including humans, in addition to exotic and rare diseases such as ebola haemorrhagic fever can also spread across the world due to the ease of global travel. Microorganisms as disease agents

7. Some early history of microbiology Brock 12 th ed. Table 1.1

8. Some early history of microbiology (a) Drawing of a bluish-coloured mold by Robert Hooke. This is the first drawing describing a microorganism. (b) Antoni van Leeuwenhoek’s drawings of bacteria. Rod-shaped: A,C,F and G; spherical or coccus-shaped: E; cocci packets: H. (c) Photomicrograph of a blood smear taken through a van Leeuwenhoek microscope. Red blood cells are visible and a single cell is about 6 μm in diameter.

9. Some early history of microbiology Ferdinand Cohn’s drawing of the large filamentous sulfur-oxidizing bacterium Beggiatoa mirabilis.

10. Robert Koch’s Germ Theory of Disease It was recognized in the 16 th century that diseases would spread in populations. Concept of contagious disease understood. Work by Pasteur (1864) and subsequent discovery of sanitization (Lister 1867) was indirect evidence that microorganisms were responsible for disease – spontaneous generation theory finally quashed. Robert Koch provided firm proof that microbes were responsible for certain diseases. "Bring out your dead," from scene 2 of the film, Monty Python and the Holy Grail http://www.microbiologyplace.com http://www.microbiologyplace.com – Spontaneous generation theory quashed.

11. Steam forced out open end Nonsterile liquid poured into flask Neck of flask drawn out in flame Liquid sterilized by extensive heating Dust and microorganisms trapped in bend Open end Long time Liquid cooled slowly Liquid remains sterile indefinitely Short time Flask tipped so microorganism-laden dust contacts sterile liquid Microorganisms grow in liquid

12. Earlier developments made Koch’s work possible Development of the light microscope. Development of conditions for growth of microorganisms in pure culture on solid media. Prevention of contamination (Ferdinand Cohn) Animal models of disease.

13. Robert Koch (1843-1910) 1862: studies medicine at University of Göttingen, Germany. He studied anthrax, a disease of the cattle and occasionally humans, caused by the endospore forming bacterium Bacillus anthracis. He noticed by careful microscopy that blood of diseased animals contained the bacterium. This association does not prove that anthrax caused the disease – maybe growth of the bacterium is a result of the disease? During his “spare time” he worked at home inoculating healthy mice with the “unpurified” anthrax from (blood of) spleens of diseased (infected) farm animals -the newly infected mice died of anthrax. He then isolated the anthrax bacillus and showed this organism still caused anthrax. He thus proved that it was the anthrax bacterium and not the “bad” blood that caused the disease.

14. KOCH’S POSTULATES The Postulates:Tools: 1. The suspected pathogenic organism should be present in all cases of the disease and absent from healthy animals. 2. The suspected organism should be grown in pure culture. Microscopy, staining Laboratory culture Red blood cells Colonies of suspected pathogen Suspected pathogen Streak agar plate with sample from either diseased or healthy animal Inoculate healthy animal with cells of suspected pathogen Observe blood/tissue under the microscope Diseased animal Healthy animal Red blood cell No organisms present

15. KOCH’S POSTULATES The Postulates: Tools: 3. Cells from a pure culture of the suspected organism should cause disease in a healthy animal. 4. The organism should be reisolated and shown to be the same as the original. Experimental animal Laboratory reisolation Diseased animal Suspected pathogen Remove blood or tissue sample and observe by microscopy Laboratory culture Pure culture (must be same organism as before) Diseased animal Healthy animal

16. Robert Koch’s work on Tuberculosis (TB) In 1881, 1/7 th of all human deaths were caused by TB – a wasting disease (consumption) Until Koch’s work, the causal agent had not been identified. He used microscopy, purified culture, tissue staining and animal inoculation. Koch developed a specific stain for Mycobacterium tuberculosis cells. Importantly he developed conditions for growth of M. tuberculosis – blood serum agar. He used Guinea pigs to examine infection (animal model). Again he satisfied his four criteria that M. tuberculosis caused TB.

17. Koch’s work on TB Koch used all his methods as he set out to identify the causative agent of TB. –Microscopy –Staining –Pure culture –Animal models Growth of M. tuberculosis in pure culture was very challenging – but he never gave up! Once pure culture growth was achieved his four postulates could be readily satisfied.

18. Studies of Mycobacterium tuberculosis (a) Section through a tubercle from lung tissue. Cells of M. tuberculosis stain blue, whereas the lung tissue stains brown. (b) Cells of M. tuberculosis in a sputum sample of a tuberculous patient. (c, d) Growth of M. tuberculosis in pure culture. (c) Growth of M. tuberculosis on a glass plate of coagulated blood serum inside a glass box (with lid open). (d) A colony of M. tuberculosis cells taken from the plate in (c) and observed microscopically at 700×; cells appear as long "cordlike" forms. Brock 12 th ed. Fig. 1.16

19. Robert Koch (1843-1910) 1882: Koch publishes his work on tuberculosis. Studied cholera in India. Did further work on pure cultures on solid media in flat dishes designed by Petri. 1905: Awarded Nobel Prize in Physiology or medicine for his decisive work on tuberculosis. http://nobelprize.org/medicine/laureates/1905/koch-bio.html

20. Mycobacterium tuberculosis Gram positive rod shaped bacterium. Grows very slowly. Cell wall has high lipid content. Primary infection in lung but can spread to other tissues. Increasing prevalence today due to AIDS and antibiotic resistance. Genome sequenced See Brock Chapter 34.5

21. Tuberculosis X-rays. (a) Normal chest X-ray. (b) Advanced case of pulmonary TB chest X-ray. Diagnosis of Tuberculosis today

22. A century later: Peptic Ulcers in the 1980’s

23. Peptic Ulcers in the 1980’s Prevailing view (pharmaceutical industry vested interest?): “ulcers were caused by acid, lifestyle stress, spicy foods, and should be treated by drugs blocking acid production” Tagamet™ and Zantac™ two of best ever selling drugs (Histamine H2 receptor blockers). These drugs did not “cure” disease but managed it - so perfect drugs for maximum profit! At the time the stomach was considered sterile -acid environment (pH 2) Barry Marshall

24. Dr. Barry J. Marshall and Dr J. Robin Warren Warren was convinced that he could visualize bacteria in stomach ulcer biopsies (Koch’s postulate number 1) Marshall developed this work. Hypothesis – “bacteria might be responsible for some stomach ulcers” However – it was difficult to grow these bacteria in the lab (just like M. tuberculosis) Growth of pure cultures (Koch’s postulate number 2) was achieved only when cultures incubated accidentally for longer than normal. Endoscopy of stomach

25. Dr Marshall was originally ridiculed for his idea He failed to infect a pig animal model (Koch’s 3 rd postulate failed). 1998 quoted as saying: "Everyone was against me, but I knew I was right". Marshall then took the rather drastic step of swallowing the purified bacterium (Koch’s 3 rd ?) His wife, Adrienne, was not impressed but she thought it typical of him! He soon became very ill. He was eventually cured by use of antibiotics. Did not even attempt Koch’s postulate number 4 (re-isolation from himself)

27. Helicobacter in the stomach mucosa Stomach protected by gastric juices by a thick mucus lining Helicobacter neutralizes acid by excreting a urease. Immune system is activated but cannot reach infection Inflammation leads to an ulcer

28. Helicobacter pylori Gram negative, highly motile, spiral shaped bacterium. Size 2.5-3.5  m long and 0.5-1  m in diameter 80% of gastric ulcer patients are infected Also implicated in gastric cancers

29. Koch’s postulates are as relevant today as ever Press Release: The 2005 Nobel Prize in Physiology or Medicine 3 October 2005The Nobel Prize in Physiology or Medicine for 2005 jointly to Barry J. Marshall and J. Robin Warren for their discovery of "the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease"

30. Further reading Brock, Chapter 1, Work of Robert Koch and work of Louis Pasteur (spontaneous generation, pasteurization, swan necked flask) http://nobelprize.org/medicine/laureates/1905/press.html http://nobelprize.org/medicine/laureates/2005/press.html