MLS1004SEF-Week 5_12 Feb 2024.pdf

Lecture 5 Introduction to Medical Microbiology and Virology I MLS 1004SEF Introduction to Medical Laboratory Science and Laboratory Instrumentation II By Dr. Andy YY CHEUNG [email protected] Book references Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology. Brooks, G....

Lecture 5 Introduction to Medical Microbiology and Virology I MLS 1004SEF Introduction to Medical Laboratory Science and Laboratory Instrumentation II By Dr. Andy YY CHEUNG [email protected] Book references Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology. Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. Mahon, C. R., & Lehman, D. C., (2019). Textbook of diagnostic microbiology. Elsevier. What is Microbiology? It is derived from Greek words: Mikros – which means small Bios – which means life Logos – which means study of Includes the study of certain nonliving entities as well as certain living organisms collectively called “microbes” Basically defined as the study of microbes Microbiology is the study of those living organisms that are not visible to the naked eye and can be seen only under the microscope (too small to be seen without magnification) Taxonomy: Classifying, Naming, and Identifying Microorganisms Taxonomy- classifying living things Microbial nomenclature- naming microorganisms Identification- discovering and recording the traits of organisms so they can be named and classified Prokaryotae (Monera) The Origin and Evolution of Microorganisms Phylogeny- the degree of relatedness between groups of living things Based on the process of evolutionhereditary information in living things changes gradually through time; these changes result in structural and functional changes through many generations Two preconceptions: All new species originate from preexisting species Closely related organisms have similar features because they evolved from a common ancestor Phylogeny usually represented by a treeshowing the divergent nature of evolution Microbial nomenclature - Assigning Specific Names The binomial system of nomenclature The generic (genus) name followed by the species name Generic part is capitalized, species is lowercase Both are italicized Staphylococcus aureus Microbial nomenclature - Assigning Specific Names Kingdom, phylum, class, order, and suborder begin with a capital letter but are not italicized If a generic plural for an organism exists, it is neither capitalized nor italicized Binary genus-species combinations are always used in the singular Genus used alone (capitalized and italicized) is usually used in the singular, but it may be used in the plural (not italicized) if it refers to all species within that genus https://wwwnc.cdc.gov/eid/page/scientific-nomenclature Scientific Nomenclature - CDC Italics are used for bacterial and viral taxa at the level of family and below Italicize species, variety or subspecies, and genus when used in the singular; Do not italicize or capitalize genus name when used in the plural Listeria monocytogenes is …listeria are; salmonellae; mycobacteria For an article about several species of the same genus, the genus must be spelled out only in the title and at first use in the abstract, text, tables, and figures; It may subsequently be abbreviated for other species We studied Pseudomonas aeruginosa, P. putida, and P. fluorescens https://wwwnc.cdc.gov/eid/page/scientific-nomenclature Virus nomenclature- Assigning Specific Names A virus is not a species; a virus belongs to a species Italicize species, genus, and family of a virus when used in a taxonomic sense it is fine to not mention taxonomy of a virus, especially one like dengue or polio that is well known Do not italicize a virus name when used generically If you capitalize a virus name (other than one that has a proper name in it so that you must capitalize it), then you need to italicize it It is permissible to use an acronym for a virus (e.g., HIV for human immunodeficiency virus), after defining it However, do not abbreviate a species e.g. Human immunodeficiency virus i.e. in short, if you do italicize, don’t use an acronym https://wwwnc.cdc.gov/eid/page/scientific-nomenclature 1. Microorganism- Eubacteria are prokaryotes are relatively simple, single-celled (unicellular) organisms Reproduce asexually (binary fission) lack nuclei much smaller than eukaryotes it is enclosed in cell walls that are largely composed of a carbohydrate and protein complex called peptidoglycan Found everywhere there is sufficient moisture; some found in extreme environments 2. Microorganism- Archaea are also prokaryotes Often found in extreme environments Not evident to cause diseases in humans Divided into 3 main groups: Methanogens – produce methane as a metabolic byproduct Extreme halophiles – live in extremely salty environments Extreme thermophiles – live in hot sulfurous water, such as hot springs 3. Microorganism- Protozoa (singular: protozoan) unicellular eukaryotes comes in variety of shapes and lives as free entities or as parasites (organisms that derive nutrient from living hosts) Similar to animals in nutrient needs and cellular structure Asexual (most) and sexual reproduction Most are capable of locomotion by Pseudopodia – cell extensions that flow in direction of travel Cilia – numerous, short, hairlike protrusions that propel organisms through environment Flagella – extensions of a cell that are fewer, longer, and more whiplike than cilia https://www.youtube.com/ watch?v=RyQfvxH425Q https://www.youtube.com/ watch?v=t6DSmymMjoA https://www.youtube.com/ watch?v=7pR7TNzJ_pA 4. Microorganism- Algae (singular: alga) are photosynthetic eukaryotes need light, water and carbon dioxide for food production and growth by photosynthesis, algae produce oxygen and carbohydrates Considered nonpathogenic to human (yet, their toxin may cause illness) abundant in fresh and salt water, in soil, and in association with plants Unicellular or multicellular Sexual or asexual reproduction Categorized on the basis of pigmentation, storage products, and composition of cell wall 5. Fungi (singular: fungus) are eukaryotes have a distinct nucleus containing genetic material (DNA) can be unicellular (yeast) or multicellular (mold) Molds – multicellular; have hyphae; reproduce by sexual and asexual spores Yeasts – unicellular; reproduce asexually by budding; some produce sexual spores Heterotrophs: obtain their food by absorbing dissolved molecules, typically by secreting digestive enzymes into their environment Possess cell walls (made of a chitin-glucan complex) 6. Multicellular animal parasites-Helminths Not strictly microorganisms Animals are eukaryotes They are of medical importance 7. Intracellular parasite- Viruses They are so small that most can be seen only with an electron microscope (except poxviruses) They are not considered living Not independently living cellular organisms Much simpler than cells- basically a small amount of DNA or RNA wrapped in protein and sometimes by a lipid membrane Individuals are called a virus particle or virion In order to reproduce, they need to use the cellular machinery of other organisms Depend on the infected cell’s machinery to multiply and disperse They are of medical importance The Golden Age of Microbiology Koch’s Postulates As originally stated, the four criteria are: (1) The microorganism must be found in diseased but not healthy individuals; (2) The microorganism must be cultured from the diseased individual; (3) Inoculation of a healthy individual with the cultured microorganism must recapitulated the disease; and (4) The microorganism must be re-isolated from the inoculated, diseased individual and matched to the original microorganism Koch’s postulates have been critically important in establishing the criteria whereby the scientific community agrees that a microorganism causes a disease Koch’s Postulates and its limitations 1. 1a. Opportunistic pathogens typically do not cause disease in healthy individuals with a properly functioning immune system; however, when the immune system is compromised, they take advantage of the opportunity to cause infection or disease 1b. Asymptomatic carrier 2. Not all pathogens are culturable 3. 3a. Some individuals have a stronger immune system 3b. Ethical issue e.g. obligate human pathogen 4. Pathogens may become less pathogenic when grown in vitro 5. Multifactorial diseases AND disease caused by more than one pathogen The germ theory of disease and its limitations Germ theory states that specific microscopic organisms are the cause of specific diseases “infectious disease is primarily caused by transmission of an organism from one host to another—is a gross oversimplification” “accords with the basic facts that infection without an organism is impossible and that transmissible organisms can cause disease” “but it does not explain the exceptions and anomalies” “The germ theory has become a dogma because it neglects the many other factors which have a part to play in deciding whether the host/germ/environment complex is to lead to infection” “Among these are susceptibility, genetic constitution, behavior, and socioeconomic determinants” Stewart, G. T. (1968). Limitations of the germ theory. The Lancet, 291(7551), 1077-1081. Development of microbiology Our knowledge of microbiology is now undergoing a remarkable transformation founded in the rapid technologic advances in molecular biotechnology The Human Genome Project was a multinational program that concluded in 2005 with the comprehensive sequencing of the human genome The techniques developed for this program have rapidly moved into the research and clinical laboratories, leading to microbial sequencing and revealing previously unappreciated insights about pathogenic properties of organisms, taxonomic relationships, and functional attributes of the endogenous microbial population Clearly, we are at the early stages of novel approaches to diagnostics and therapeutics based on the monitoring and manipulations of this population (the microbiome) We now know that there are thousands of different types of microbes that live in, on, and around us—and hundreds that cause serious human diseases Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Microbes and Humans We are made up of about 1 trillion cells At any point in time you have about 10 trillion microbial cells growing on or inside you Microbes and Humans microbes may be classified as cellular or acellular (viruses & prions) most are harmless, but ~3% are pathogens (cause disease) Impact of infectious diseases caused by pathogenic microbes Host-Pathogen-Environment-Microbiome interaction Schmeller, D. S., Courchamp, F., & Killeen, G. (2020). Biodiversity loss, emerging pathogens and human health risks. Biodiversity and conservation, 29, 3095-3102. The Modern Age of Microbiology How Do Genes Work? Molecular biology Recombinant DNA technology Gene therapy Microbial genetics The Modern Age of Microbiology Recombinant DNA Technology Genes in microbes, plants, and animals manipulated for practical applications Production of human blood-clotting factor by E. coli to aid hemophiliacs Gene Therapy Inserting a missing gene or repairing a defective one in humans by inserting desired gene into host cells 賀建奎 https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapiestreat-patients-sickle-cell-disease https://www.globenewswire.com/newsrelease/2022/09/15/2516733/0/en/Excision-BioTherapeuticsDoses-First-Participant-in-EBT-101-Phase-1-2-Trial-EvaluatingEBT-101-as-a-Potential-Cure-for-HIV.html The Modern Age of Microbiology How Do We Defend Against Disease? Immunology The study of the body’s defense against specific pathogens Serology The study of blood serum existence of chemicals and antibodies in the blood that fight infection Chemotherapy Antimicrobials (antibiotics, antifungals, antivirals, antiparasitics) Epidemiology (in medical microbiology) the study of the frequency, distribution, and spread of infectious diseases; it involves understanding how infections spread, identifying risk factors, and implementing strategies for disease prevention and control Medical microbiology Medical microbiology is the study of causative agents of infectious diseases of humans and their reactions to such infections It deals with etiology, pathogenesis, laboratory diagnosis, specific treatment and control of infection (e.g. immunization) Definition Terms in medical microbiology Etiology – the study of the causes or origins of diseases or conditions; It involves investigating and understanding the factors, agents, or events that contribute to the development of a particular disease or condition Pathogenesis – the process by which a disease or condition develops and progresses within the body Laboratory diagnosis – the use of various laboratory tests and techniques to aid in the identification and confirmation of a disease or medical condition Treatment in medical microbiology – the strategies and interventions used to manage and cure infectious diseases caused by microorganisms such as bacteria, viruses, fungi, and parasites; the goal of treatment is to eliminate or control the infection, alleviate symptoms, prevent complications, and promote recovery Infection control – the measures and practices implemented to prevent and reduce the transmission of infectious agents in healthcare settings, communities, and other environments Definition Terms in medical microbiology Medical Bacteriology – the science of bacteria that can cause infectious diseases in human Medical Virology – the science of viruses, nonliving entities that can cause infectious diseases in human Medical Mycology – the study of fungi that are pathogenic for human Medical Parasitology –the science that deals with organisms living in the human body and the medical significance of this host-parasite relationship Medical Protozoology – the study of protozoan parasites and the diseases they cause in humans Pathogens – microbes that cause diseases Nonpathogens – microbes that do not cause disease Commensals – microbes that reside on either surface of the body or at mucosa without harming human health Normal flora – the community of microorganisms that reside in or on the human body; these microorganisms include bacteria, fungi, viruses, and other microbes SIX causative agents (pathogens) 8. Prions abnormally folded protein that can cause a variety of neurodegenerative diseases in humans (CJD and vCJD) and animals ("mad cow disease“ in cattle) Unlike bacteria or viruses, prions do not contain genetic material like DNA or RNA convert normal proteins into their abnormal form, leading to the accumulation of these misfolded proteins in the brain and other tissues This accumulation disrupts normal cellular function and causes progressive damage to the nervous system 8. Prions Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are characterized by the accumulation of prions in the brain, resulting in the formation of microscopic holes and a sponge-like appearance under a microscope Highly resistant to usual disinfection and sterilisation methods The vast majority of CJD patients usually die within 1 year of illness onset Possibility of transmission Acquired CJD: Through transplantation Acquired CJD : Through instruments contaminated with brain tissue, dura or cerebrospinal fluid of infected person vCJD: via ingestion of cattle products contaminated with the prions Comparison among different causative agents Chlamydia Rickettsiae How about Helminths? The Five “I”s in microbiology Inoculation: to introduce microbes into a culture media so that it reproduces there Isolation: to obtain pure culture Incubation: to grow microbes under proper conditions Inspection: to observe characteristics of microbes Identification: to ID organism to exact species level Microscopy In general, microscopy is used in microbiology for two basic purposes: the initial detection of microbes and the preliminary or definitive identification of microbes bacterial cells, fungal elements, parasites (eggs, larvae, or adult forms), and viral inclusions present in infected cells For examples, by characteristic morphologic properties: preliminary identification of most bacteria definitive identification of many fungi and parasites With antibodies labeled with fluorescent dyes or other specific identification of organisms in clinical specimens Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Microscopy The Light Microscope Resolving power: about half the wavelength of the light being used Resolving power is the distance that must separate two point sources of light if they are to be seen as two distinct images. With light of a wavelength of 0.4 mm (smallest in visible light), the smallest separable diameters are thus about 0.2 mm (i.e. 1/3 width of a typical prokaryotic cell) Useful magnification of a microscope is the magnification that makes visible the smallest resolvable particles. Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. Light Microscope Image source: https://microbenotes.com/light-microscope/ Observation with a light microscope (low-power field and high-power field) https://www.youtube.com/watch?v=QhjT6_4zDws Observation with a light microscope (low-power field and high-power field) https://www.youtube.com/watch?v=SUo2fHZaZCU Observation with a light microscope (Oil Immersion Microscopy; Oil lens field) https://www.youtube.com/watch?v=zzamomqlwxU Several types of light microscopes are commonly used in microbiology: A. Bright-Field Microscope consists of two series of lenses (objective and ocular lens), which function together to resolve the image a 100-power objective lens with a 10-power ocular lens magnifies the specimen 1000 times Particles 0.2 µm in diameter: magnified to about 0.2 mm and so become visible Further magnification: give no greater resolution of detail and reduce the visible area (field) With this microscope, specimens are rendered visible because of the differences in contrast between them and the surrounding medium. Bacteria are difficult to see: lack of color and contrast with the surrounding medium Dyes (stains): increase the contrast, bacteria becomes more easily seen Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. Several types of light microscopes are commonly used in microbiology: B. Dark-Field Microscope lighting system modified to reach the specimen from the sides only accomplished by the use of a special condenser that both blocks direct light rays and deflects light off a mirror on the side of the condenser at an oblique angle creates a “dark field” that contrasts against the highlighted edge of the specimens and results when the oblique rays are reflected from the edge of the specimen upward into the objective of the microscope Higher resolution useful to observe organisms that is smaller than 0.2 µm in diameter e.g. Treponema pallidum , a spirochete Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. Image source: https://microscopewiki.com/brightfield-vs-darkfield-microscope/ Darkfield Microscopy Tutorial https://www.youtube.com/watch?v=Tw557w421is Treponema pallidum by Dark-Field Microscope https://phil.cdc.gov/details.aspx?pid=2335 Several types of light microscopes are commonly used in microbiology: C. Fluorescence Microscope The fluorescence microscope is used to visualize specimens that fluoresce , which is the ability to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible) Some organisms fluoresce naturally because of naturally fluorescent substances e.g. chlorophyll Those that do not naturally fluoresce may be stained with fluorochrome (fluorescent dye) Fluorescence microscopy is widely used in clinical diagnostic microbiology. Fluorescent-antibody (FA) technique or immunofluorescence. For example, the fluorochrome auramine O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis, the bacterium that causes tuberculosis. When the dye is applied to a specimen suspected of containing M tuberculosis and exposed to ultraviolet light, the bacterium can be detected by the appearance of bright yellow organisms against a dark background. The fluorescent treponemal antibody absorption (FTA-ABS) test Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. Diagnosis of active tuberculosis disease: From microscopy to molecular techniques https://phil.cdc.gov/PHIL_Images/20040 615/238aba2af0c347f7a0f744ff52b7c10 d/5790_lores.jpg https://phil.cdc.gov/PHIL_Images/20041202/1a 0f953285f84cd4b3d1b31237a7a5a8/6468_lores.jpg Caulfield, A. J., & Wengenack, N. L. (2016). Diagnosis of active tuberculosis disease: From microscopy to molecular techniques. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases, 4, 3343. Fluorescent treponemal antibody absorption (FTA-ABS) test Image source: https://microbeonline.com/fluorescent-treponemal-antibody-absorption-fta-abs-test/ Besides light microscopes, electron microscopes are also used in microbiology: D. Electron Microscope High resolving power: observe the detailed structures of microorganisms Superior resolution of the electron microscope is due to the fact that electrons have a much shorter wavelength than the photons of white light Two types of electron microscopes: the transmission electron microscope (TEM), which has many features in common with the light microscope, and the scanning electron microscope (SEM) TEM uses a beam of electrons projected from an electron gun and directed or focused by an electromagnetic condenser lens onto a thin specimen electrons strike the specimen, they are differentially scattered by the number and mass of atoms in the specimen some electrons pass through the specimen and are gathered and focused by an electromagnetic objective lens, which presents an image of the specimen to the projector lens system for further enlargement When the electrons struck a screen, it fluoresces and image is visualized Image visualized by a fluorescent screen, a layer of photographic film, or a sensor attached to a charge-coupled device TEM can resolve particles 0.001 µm apart; Viruses with diameters of 0.01–0.2 µm can be resolved Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. Transmission Electron Microscope Image source: https://www.cambridge.org/core/books/analytical-geomicrobiology/applications-of-transmissionelectron-microscopy-in-geomicrobiology/04F139754543AF817BAEAC6DB9608B29 Image source: https://www.hitachi-hightech.com/global/science/products/microscopes/electronmicroscope/tem/ht7800.html Compare and contrast Light and Electron Microscope Virus size comparison https://viralzone.expasy.org/5216 Can you see a virus with the light microscope? No, except poxviruses. Microscopy- Direct Examination Direct examination methods are the simplest for preparing samples for microscopic examination Wet mount: suspended in water or saline KOH method: mixed with alkali (e.g. potassium hydroxide) to dissolve background material Or, mixed with a combination of alkali and a contrasting dye The dyes nonspecifically stain the cellular material, increasing the contrast with the background, and permit examination of the detailed structures Negative staining method: India ink darkens the background rather than the cell to detect capsules surrounding organisms, such as the yeast Cryptococcus and encapsulated Bacillus anthracis Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Microscopy- Direct Examination Wet mount of Trichomonas vaginalis https://www.youtube.com/watch?v=rim2dXF3Oac KOH test of fungi India ink method Image source: https://microbeonline.com/cryptococcusneoformans-properties-pathogenesis-diseases-lab-diagnosis/ Image source: https://universe84a.com/koh-test/ Microscopy- Differential Stains A variety of differential stains are used to stain specific organisms or components of cellular material Gram stain: best known and most widely used stain Bacteria and yeasts (yeasts are gram-positive) Acid fast stain Differentiate acid fast bacteria (e.g. mycobacterium) from non-acid fast bacteria Iron hematoxylin and trichrome stains: protozoan parasites Wright-Giemsa stain: blood parasites Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Gram staining technique https://www.youtube.com/watch?v=sxa46xKfIOY Gram staining technique https://www.youtube.com/watch?v=--OvDvS-Pec Microscopy- Differential Stains A variety of differential stains are used to stain specific organisms or components of cellular material Gram stain: best known and most widely used stain Bacteria and yeasts (yeasts are gram-positive) Image source: https://www.quora.com/What-are-the-mechanismsof-gram-staining-and-how-do-they-work Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Inspection of bacteria Microscopic examination: It helps to detect a shape, a size and an arrangement of microorganisms Staining reaction: On gram staining we can have two groups of microorganisms: Gram positive and Gram negative A. E. coli, Gram negative, B. Staphylococcus epidermidis, Gram positive C. Bacillus cereus, Gram positive Acetone Alcohol (50% Acetone / 50% Alcohol) Morphology of Bacteria Lecture 6 Introduction to Medical Microbiology II -Colony morphology SCI 8007SEF Medical Microbiology & Virology By Dr. Andy YY CHEUNG [email protected] Supplementary material-Colonial Characteristics by US CDC (Division of Laboratory System) Colony morphology (Please read before Laboratory session)