Introduction to Medical Microbiology PDF

Chapter 1 Introduction to Microbiology Contact Details Dra Veronica Veses – [email protected] – Room 324, 3rd Floor, Health Sciences Building Dr Chirag Sheth – [email protected] – Room 343, 3rd Floor, Health Sciences Building Dra Antonella Locascio...

Chapter 1 Introduction to Microbiology Contact Details Dra Veronica Veses – [email protected] – Room 324, 3rd Floor, Health Sciences Building Dr Chirag Sheth – [email protected] – Room 343, 3rd Floor, Health Sciences Building Dra Antonella Locascio – [email protected] – Room 322, 3rd Floor, Health Sciences Building Dr Slaven Erceg – [email protected] – Room 344, 3rd Floor, Health Sciences Buiding 2 Course Sections MICROBIOLOGY - Basic principles of medical microbiology - General principles of laboratory diagnosis - Bacteriology - Virology - Mycology - Parasitology 3 Lectures Topic 1. Introduction to Medical Microbiology. Topic 2. Microbial growth and control. Topic 3. Antimicrobial agents. Topic 4. Host-pathogen relationships in microbial infections. Topic 5. Diagnostic microbiology. Topic 6. Coccus: Staphylococci, Streptococci, Enterococci, Neisseria Topic 7. Gram positive Bacilli: Bacillus, Corynebacterium and Listeria, Clostridium Topic 8. Gram negative Bacilli: Enterobacteriaceae, Vibrio, Pseudomonas, Campylobacter and Helicobacter Topic 9. Cocobacilli and Mycobacteria: Haemophilus, Bordetella, Brucella, Francisella, Legionella, Mycobacterium and Nocardia. Topic 10. Spirochaetes and Mycoplasmas: Treponema, Borrelia, and Leptospira, Mycoplasma and Ureaplasma. Topic 11. Intracellular bacteria: Rickettsia, Orientia, Ehrlichia, Anaplasma, Coxiella, and Chlamydiaceae Topic 12. Viruses and other subcellular agents. Topic 13. Clinical Virology: DNA and RNA Virus. Topic 14. Frequent infections by fungal agents. Topic 15. Frequent infections by parasitic agents. 4 References Textbooks – MEDICAL MICROBIOLOGY 7TH EDITION, Patrick R Murray; Ken S Rosenthal; Michael A Pfaller, Editorial Philadelphia: Mosby/Elsevier, 2013 – PRACTICAL MEDICAL MICROBIOLOGY FOR CLINICIANS, Frank E. Berkowitz, Robert C. Jerris, Editorial : Wiley- Blackwell, 2016 5 What is Microbiology? Microbiology studies small (micro) living organisms and covers a wide series of topics such as their physiology, ecology, taxonomy. This subject will focus on the impact of microorganisms in human health and the consequences of a microbial infection. 6 HISTORY OF MICROBIOLOGY 7 1590: Invention of the Microscope Three Dutch spectacle makers—Hans Jansen, his son Zacharias Jansen, and Hans Lippershey— have received credit for inventing the microscope about 1590. It was a compound microscope, with an eyepiece and an objective lens, made of wood and cardboard 8  1665: Robert Hooke He looked at a sliver of cork through a microscope lens and discovered cells. He also wrote a book (Micrographia) describing fleas, roots, head lice, insects, and many others 9 1677: Microorganisms were first observed by Anton van Leeuwenhoek, the “Father of Microbiology” He first observed “animalcules” (single- cell organisms, called protozoa) from his teeth scrapings, after improving the microscope. He discovered bacteria, protozoa, sperm and blood cells, etc. 10  Lens Specimen holder Focus screw Handle 11 1796: Edward Jenner, pioneer of vaccination and Father of Immunology Edward Jenner, an English doctor, performed his famous experiment on an 8-year old boy. He took the pus from a cowpox pustule and injected it into the boy- based on the popular tale that milkmaids developed a mild form of the disease once, but then they never contract the cowpox themselves. Jenner subsequently proved that after having been inoculated with the pus from a cowpox pustule, the boy was now immune to smallpox. Furthermore, Jenner experimented on several other children, including his 11-month-old son and finally in 1798, the results were published and Jenner coined the word vaccine from the Latin ‘Vacca’ for the cow. 12 13 1846: Ignaz Semmelweis He was a hungarian doctor, who observed differences in maternal mortality after births given by doctors and midwives. The main difference were that doctors were performing autopsies before assisting the births. He implemented mandatory hand washing and chlorine desinfection of instruments 14 1864: Louis Pasteur invents Pasteurization Louis Pasteur observed that to prevent spoilage in beer, alcohols, and milk heating them was sufficient to kill the pathogenic microbes at a specific temperature and for a specific time without changing the taste of the drinks. 15 1867: Sir Joseph Lister Sir Joseph Lister, pioneer of antiseptic surgery, applied Louis Pasteur’s principles and taking a note of his advances, introduced phenol, otherwise, earlier known as carbolic acid to sterilize surgical instruments, post and pre- surgery and to clean wounds to reduce post- surgical infections 16 1876: Robert Koch Robert Koch provided the much needed first proof on the Germ Theory by working on B. anthrax. The procedure he followed was then postulated into Koch’s Postulates and it helped proved that microbes are the causative agent of any given infection or disease. 17 Koch’s Postulates Postulate 1: The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms. Postulate 2: The microorganism must be isolated from a diseased organism and grown in pure culture. Postulate 3: The cultured microorganism should cause disease when introduced into a healthy organism. Postulate 4: The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent. 18 1892: Dmitri Ivanovski: foundation of Virology Ivanoski reported that extracts taken from infected tobacco leaves were still infectious after rigorous filtration. He assumed that it is either the bacteria that were retained and filtered or it must be the filterable bacterial toxins that are active and cause infections. This was the basis or the foundation step for the field of Virology. 19 1910: Paul Ehrlich invents the concept of chemotherapy 1928: Sir Alexander Fleming discovers penicillin Paul Ehrlich Sir Alexander discovered Fleming discover Salvarsan, an arsenic penicillin, a derivative useful to substance produced treat syphilis by Penicillium notatum (a fungi) with antibacterial properties 20 1931-38: Max Knoll and Ernst Ruska developed the prototype of the electron microscope 21 ROLE OF MICROORGANISMS 22 Importance of Microbiology Basic Science: Theory of Evolution – bacterial origins Agriculture: Photosynthesis and decomposition Food Microbiology: Human uses of microorganisms Public Health and Epidemiology: Infectious diseases Healthy Microbiome 23 Bacteria appeared approximately 3.5 billion years ago 24 Microbes are involved in photosynthesis - account for >50% of the earth’s oxygen. Decomposition – nutrient recycling. 25 Microbes are used in food industry, to synthesize medicines and vaccines, and bioremediate contaminated water 26 Microbes are responsible for diseases in humans and animals Perhaps one of the most important observations in clinical microbiology clearly defining the link between microorganisms and disease Robert Koch verified the germ theory, which states that many diseases are caused by microorganisms and not by sins, bad character, poverty or other social problems 27 Microorganisms and infectious disease 28 29 What are the main differences between countries with respect to causes of death? vaccination programs antibiotics 30 2020: CORONAVIRUS PANDEMIC Epidemique or endemic (differnces)  pandemic In 2019 a novel coronavirus emerges from animals to humans causing an outbreak in Wuhan (China), that becomes epidemic, and then pandemic. 31 32 HEALTHY MICROBIOME 33 The human microbiome Up until the time of birth, the human fetus lives in a remarkably protected and for the most part sterile environment; however, this rapidly changes as the infant is exposed to bacteria, archaea, fungi, and viruses from the mother, other close contacts, and the environment. Over the next few years, communities of organisms (microbiota or normal flora) form on the surfaces of the skin, nose, oral cavity, intestines, and genitourinary tract. Most individuals share a core microbiome, defined as the species that are present at a specific site in 95% or more of individuals. The greatest numbers of shared species are present in the mouth, followed by the nose, intestine, and skin, and the fewest shared species are found in the vagina. 34 35 Microorganisms affect ALL aspects of our existence… recently named the “the last organ” 36 Functions of core microbiome The host provides a place to colonize, nutrients, and some protection from unwanted species (innate immune responses). The microbes provide: – needed metabolic functions, – stimulate innate and regulatory immunity, – prevent colonization with unwanted pathogens. If the microbiota gets unbalanced a dysbiosis is produced. This leads to chronic inflamation and potentially cancer. 37 THE MICROBIAL WORLD 38  The tree of life: Woese’s Three Domain Classification System If you remove fungi, plants, ciliates all the rest is microbiology 39 Domains The domains are clustered on the basis of 16S rRNA sequence and are further divided into: – Kingdom – Phylum – Class – Order – Family – Genus – Species 40 There are four main types of microorganisms in Medical Microbiology Bacteria Fungi Parasites Virus 41 Bacteria This domain includes a group of phylogenetically related prokaryotes distinct from Archaea It is estimated that bacterial species on Earth number in the hundreds of thousands, of which only about 5500 have been discovered and described in detail. 42 Bacteria Bacteria are prokaryotes (lacking a formal nuclear structure) Their DNA is usually a single molecule, generally covalently closed and circular (bacterial chromosome) Sometimes they carry extra-chromosomal DNA (plasmids) Bacterial cells are smaller than eukaryotic cells, usually less than 2 micrometres in diameter Responsible for many serious human and animal infections (syphilis, faringitis, endocarditis, anthrax,…) 43 Escherichia coli Enterococcus 44 Fungi 2° more common in the word after insect Fungi are everywhere. There are approximately 1.5 million different species of fungi on Earth, but only about 300 of those are known to cause disease in humans, Fungi are eukaryotes They live outdoors in soil and on plants and trees as well as on many indoor surfaces and on human skin. – Mild fungal skin diseases can look like a rash and are very common. – Fungal diseases in the lungs are often similar to other illnesses such as the flu or tuberculosis. – Some fungal diseases like fungal meningitis and bloodstream infections are less common than skin and lung infections but can be deadly. 45  Aspergillus Candidiasis in the throat Associate with diabetes https://www.cdc.gov/fungal/diseases/index.html 46 Parasites A parasite is an organism that lives on or in a host and gets its food from or at the expense of its host. There are three main classes of parasites that can cause disease in humans: protozoa, helminths, and ectoparasites. 47 Types of parasites Protozoa are microscopic, one-celled organisms that can be free-living or parasitic in nature. They are able to multiply in humans, which contributes to their survival and also permits serious infections to develop from just a single organism. Helminths are large, multicellular organisms that are generally visible to the naked eye in their adult stages. Like protozoa, helminths can be either free-living or parasitic in nature. In their adult form, helminths cannot multiply in humans. Ectoparasites: ticks, fleas, lice, and mites that attach or burrow into the skin and remain there for relatively long periods of time (e.g., weeks to months). 48 Plasmodium falciparum in blood Ascaris lumbricoides https://www.cdc.gov/parasites/about.html 49 Virus Viruses are genetic elements that can replicate independently of a cell’s chromosomes but not independently of cells themselves Sub microscopic entities consisting of a single nucleic acid surrounded by a protein coat and capable of replication only within the living cells of bacteria, animals or plants Obligate Intracellular Parasites Viruses are characterized by having an extracellular state, known as virion, in this extracellular state the virus particle is metabolically inert and does not carry out respiratory or biosynthetic functions 50 Hepatitis C virus HIV 51 Origin of viruses The origin of modern viruses is unclear. There are two hypotheses: They could be fugitive pieces of nucleic acid belonging to a larger body that broke off and became active, therefore, new viruses are being formed frequently and many do not have ancestors The viruses once lived outside the host cells, but with time due to its parasitic lifestyle, lost the genes required to live outside the host 52 Virion structure DNA or RnA viruses but not both Envellope virus (prot) vs naked virus (prot + lipidic coat) The structure of virions is quite diverse, presenting differences in size, shape, and chemical composition. The nucleic acid of the virion is always located within the particle, surrounded by a protein coat called the capsid (coat, shell). The protein coat is always formed of a number of individual protein molecules, called structural subunits, which associate to form morphological units or capsomers. The complete complex of nucleic acid and protein is called the virus nucleocapsid. If the nucleocapsid is enclosed in a membrane the virus is enveloped. Otherwise is called naked virus. 53 Virion structure 54 55  Depend on: Virus classification Species that infect: animal viruses, plants, bacteria,... Presence or absence of lipid envelope Symmetry of the nucleocapsid Depending on the type of nucleic acid (DNA or RNA) Depending on the number of nucleic acid strands, their structure and the polarity of the viral genome. 56 TAXONOMY 57 Taxonomy A system for organising, classifying and naming living organisms. The primary concerns of taxonomy are classification and nomenclature. Modern biologists currently use the three-domain (archaea, bacteria and eukarya) taxonomic system developed by Dr. Carl Woese in 1990. 58 Domain: Bacteria The domains are classified in phyla, classes, orders, families, genera, and species, plus subtypes if any. For example: Phylum Proteobacteria Class Gamma proteobacteria Order Coccidioides Family Enterobacteriaceae Genus Escherichia Species E. coli Subtype Serovar O157 59 Species The “basic unit” of taxonomy, representing a specific, recognized type of organism is the SPECIES. For sexually reproducing organisms, a fundamental definition of “species” has been reproductive compatibility Organisms within a genus generally share 93% similar rRNA Organisms within a species generally share 97% similar rRNA This definition fails for many microbial species (including bacteria), because they do not reproduce sexually. 60 Species in non-sexually dividing organisms A population of cells with similar characteristics – Clone: A population of cells derived from a single cell (genetically identical) – Strain: A subgroup within a species with one or more characteristics that distinguish it from other subgroups in the species (not genetically identical) 61 Binomial system of nomenclature Scientific or Systematic Name: Genus name + species name Italicized or underlined Genus name is capitalized and may be abbreviated Species name is never abbreviated A genus name may be used alone to indicate a genus group; a species name is never used alone example: Bacillus subtilis B. subtilis 62 Examples Scientific binomial Genus name Species name Klebsiella Honors Edwin Klebs The disease pneumoniae Francisella tularensis Honors Edward Tulare (California) Francis Streptococcus Chains of cells Forms pus (pyo-) pyogenes (strepto-) Trypanosoma cruzi corkscrew-like Honors Oswaldo (trypano-) soma (- Cruz body) 63  How do we name Bacteria 1. Bergey’s Manual of Determinative Morphology, differential Bacteriology staining, biochemical tests (phenotypic/biochemical). Provides identification schemes for identifying bacteria and archaea 2.Bergey’s Manual of Systematic Based on rRNA sequencing Bacteriology (genetic). Provides phylogenetic information on bacteria and archaea 3. Approved Lists of Bacterial Names Based on published articles Lists species of known prokaryotes http://www.bacterio.net/-alintro.html 64 How do we name Fungi, Parasites and Virus Fungi – Mycobank: www.mycobank.org – Index Fungorum: www.indexfungorum.org/ Parasites: – CDC: www.cdc.gov/dpdx Virus – International Committee on Taxonomy of Viruses (ICTV): https://talk.ictvonline.org/ 65 Chapter 1-Part II Bacterial classification, morphology, structure and metabolism Dra Verónica Veses-Jimenez [email protected] BACTERIAL CLASSIFICATION 67 Bacterial classification Bacteria are single celled microbes. The cell structure is simpler than that of other organisms as there is no nucleus or membrane bound organelles. Classification can be done on the basis of: – Morphology – Structure – Metabolism 68 BACTERIAL MORPHOLOGY 69 Coccus: round shape A=grape x 70  Examples Streptococcus Neisseria Gonoria = sexual transmited disese Staphylococcus 71 Bacilli: elongated, rod-shape intermediate 72 Examples Escherichia coli Bacillus anthracis 73 Other shapes Vibrio Spirillum Diaherea  risk of death Not pathology in human Vibrio cholerae Spirillum volutans 74 Other shapes: Spirochetes Siphylis =STD Treponema pallidum 75 Summary Shape of the cell is referred to as its morphology Bacteria vary in size from 0.1 to 50 micrometres 76 BACTERIAL STRUCTURE 77  Bacterial structure Intracellular structures: – Bacterial chromosome – Plasmids (not always) – Ribosome (70S): two subunits, 30S and 50S – Cell membrane (no cholesterol) Extracellular structures – Flagella – Pili – Cell wall – Capsule (not always) 78  Flagella No good if they have flagella because they move away from antibiotics long helical filaments extending from the cell surface, made (mainly) of a protein called flagellin involved in chemotaxis (positive and negative) 79 Types of flagella 80 Pili, also known as Fimbriae More stiky they are …more pathogen short, rigid and numerous involved in: – Attachment to other bacteria and/or host cells – Avoid phagocytosis and immune recognition 81 CELL WALL The main structural component of the cell wall is a peptidoglycan (also known as murein). It is a mixed polymer of hexose sugars (N- Penicelin acid) acetylglucosamine and N-acetylmuramic batheriostatic and amino acids. 82 Gram pattern In 1882, Christian Gram devised a bacterial staining method to visualize bacterial cell wall that allowed to differenciate bacteria in two major groups. Bacteria are classified according to their cell wall as Gram-positive or Gram-negative. 83  Gram positive and negative structure In Gram-positive the peptidoglycan forms a thick (20- 80 nm) layer, external to the cell membrane, and may contain other macromolecules In Gram-negative species the peptidoglycan layer is thin (5-10 nm) and is overlaid by an outer membrane, which contains lipopolysaccharides and lipoprotein The periplasm is the space between the inner and outer membrane in Gram-negative bacteria. In Gram-positive bacteria a smaller periplasmic space is found between the inner membrane and the peptidoglycan layer. 84 Lipopolysaccharide Conserved structure in all Gram-negative bacteria Essential for structural integrity and viability of the bacteria LPS is also known as endotoxin because it induces a strong immune response in the host. It has also been implicated in other aspects of bacterial ecology, such as surface adhesion. 85 LPS structure 86 Other cell wall components Teichoic acids: typically have a backbone of (polyol- phosphate)n, usually with sugars and/or the amino acid D-alanine as substituents. They are probably involved in uptake of Mg2+ by the cell. Only present in Gram positives. Teichuronic acids: similar polymers found in capsules or LPS in some Gram negative bacteria. Other: Lipids, proteins… 87 CapsuleSugar layer hyding the bacteria Capsule  meningites No capsule otites External to the cell wall there may be an additional capsule of high molecular weight polysaccharides that give a slimy surface. Very important in virulence, as protects the microorganism from the host immune system. 88 BACTERIAL NUTRITION AND METABOLISM 89 Nutrition It is a process by which chemical substances (nutrients) are acquired from the environment and used in cellular activities. Bacteria take up small molecules such as amino acids, oligosaccharides and small peptides across the cell wall. Transport of nutrients into the cytoplasm is achieved by the cell membrane using facilitated diffusion or active transport. 90  Gram-negative species can also take up larger molecules after preliminary digestion in the periplasmic space. 91 Microbial metabolism: From the Greek word metabole, meaning change. Metabolism - the sum of the biochemical reactions required for energy generation AND the use of energy to synthesize cell material from small molecules in the environment. Why is important? Because we want to know how to inhibit metabolism to control bacterial growth 92 Metabolism Two components: Anabolism - biosynthesis – building complex molecules from simple ones – requires ENERGY (ATP) Catabolism - degradation – breaking down complex molecules into simple ones – generates ENERGY (ATP) 3 Biochemical Mechanisms Utilized – Aerobic Respiration – Anaerobic Respiration – Fermentation 93 METABOLIC DIVERSITY Bacterial metabolism is classified into nutritional groups on the basis of three major criteria: Source of energy. Source of carbon. Source of electron acceptors (energy production). 94 These requirements can be combined: In every possibility there is bacterias All pathogen bacteria are in the last group chemoorganotrophes 95 All pathogenic bacteria are organotrophs They obtain energy by oxidizing preformed organic molecules (carbohydrates, lipids and proteins) from their environment 96 3. Energy Production Three Biochemical Mechanisms Utilized: Aerobic Respiration Anaerobic Respiration Fermentation 97 Aerobic Respiration Molecular Oxygen (O2) serves as the final e- acceptor – O2 is reduced to H2O 3 Coupled Pathways Utilized – Glycolysis – Kreb’s Cycle or Tricarboxylic Acid Cycle or Citric Acid Cycle – Respiratory Chain or Electron Transport Chain (ETC) 98 Anaerobic respiration Utilizes same 3 coupled pathways as Aerobic Respiration Used as an alternative to aerobic respiration Final electron acceptor something other than oxygen: NO3- : Pseudomonas, Bacillus. SO4-: Desulfovibrio CO3-: methanogens Lower production of ATP because only part of the TCA cycle and the electron transport chain operate. 99 Fermentation Incomplete oxidation of glucose or other carbohydrates in the absence of oxygen Uses organic compounds as terminal electron acceptors Effect - a small amount of ATP Production of ethyl alcohol by yeasts acting on glucose Formation of acid, gas & other products by the action of various bacteria on pyruvic acid 100 101