Introduction To Clinical Microbiology PDF

logo Clinical Microbiology and Communicable Diseases (Course code: CCST4050) Unit 1A Introduction to Clinical Microbiology Dr. Summer Xia [email protected]...

logo Clinical Microbiology and Communicable Diseases (Course code: CCST4050) Unit 1A Introduction to Clinical Microbiology Dr. Summer Xia [email protected] 1 Introduction to Clinical Microbiology Part I : What is clinical microbiology? Overview Significance Part II: What is microorganism? Classification Structures Multiplication Part III: Microbes and you Normal microbiota, pathogens, opportunistic pathogens Symbiotic relationships Part IV: Pioneers of Microbiology 2 Part I: What is Clinical Microbiology? 3  Overview Applied microbiology Environmental Clinical Microbiology Microbiology microbiology Food Microbiology 4 http://www.ibp.ethz.ch/research/environmentalmicrobiology/83742939.pidwrNCk.jpg http://www.sciencephoto.com/image/134843/530wm/C0071846-Cefalexin_antibiotic_drug-SPL.jpg http://images.sciencedaily.com/2006/10/061017091752.jpg http://www.kln.ac.lk/science/depts/micro/images/stories/microbiology/segment_microbiology.jpg Overview Basic characteristics of microorganisms 1 2 Microbial pathogenesis Clinical Microbiology 3 Host defense mechanism 4 Treatment and prevention of infections 5 Diagnosis of infections 5 What is clinical microbiology?  Study of causative agents of infectious diseases of human and their reactions of such infection  Study of microorganisms affecting health and wellness  Bacteria (bacteriology)  Fungus (mycology)  Parasites (protozoology)  Viruses (virology) 6 Why is it important to study clinical microbiology?  To understand: how microorganisms interact with individuals and populations, how microorganisms cause diseases, and how these diseases can be prevented and/or controlled.  Pathogenic microorganisms cause diseases in humans.  Microorganisms can also produce antibiotics used to treat diseases.  Microbiologists determine pathogen causing the disease and identify a substance (usually an antibiotic) to inhibit the microorganism. 7 Part II: What are microorganisms? 8 What is a microorganism?  Definition: an organism that is microscopic  Too small to be seen by the naked eyes  Most can only be seen under the microscope  Naming of microorganisms  Binomial nomenclature First name is the genus name (starting with a capital) Second name is the specific epithet (in small letters) Joining the two names together refers to a species Both names are always italicized or underlined e.g. Staphylococcus aureus or Staphylococcus aureus  After the first use, scientific names may be abbreviated with the first letter of the genus and the specific epithet: Staphylococcus aureus and Escherichia coli are found in the human body, S. aureus on skin and E. coli in the large intestine. 9 http://www.scienceprofonline.com/microbiology.html Major types of microorganisms A. Cellular microbes:  Prokaryotes (without nucleus): e.g. bacteria  Eukaryotes (with nucleus): e.g. fungi, protozoa, algae B. Acellular microbes (also called infectious particles) *acellular = not composed of cells  Viruses  Prions (infectious proteins) 10 Major types of microorganisms Microbes Cellular Acellular microbes microbes Prokaryotes Eukaryotes Viruses Bacteria Fungi Protists (protozoa & algae) 11 Major types of microorganisms  Their main characteristics: 1. Bacteria: prokaryotic - lack a cell nucleus 2. Fungi: eukaryotic - have a cell nucleus 3. Protists: eukaryotic - have a cell nucleus 4. Viruses: are not cells - they are tiny, inert particles containing DNA or RNA that must infect an appropriate host cell in order to reproduce and survive. 12 Major types of microorganisms Bacterium: E. coli Fungus: Candida albicans Protozoa: Entamoeba Virus: Influenza virus 13 histolytica Images from ebook: Lippincott’s illustrated reviews: Microbiology (3rd Ed.), Lippincott Williams & Wilkins Prokaryotes and Eukaryotes 14 Prokaryotes and Eukaryotes Prokaryotes Eukaryotes No nucleus True nucleus Lack mitochondria and Have membrane-bound chloroplasts organelles (e.g. mitochondria and chloroplasts) Simpler, smaller More complex Reproduced by binary fission Reproduced by mitosis or (simple division of one cell meiosis into two cells) 15 Bacteria  Most abundant organisms on earth  Have rigid cell wall composed of peptidoglycan (unique to bacteria)  Prokaryotes  Cell walls have characteristic  Lacking a cell nucleus shapes that are often used to help  Typically contain a single circular name the bacteria: chromosome a spherical Coccus  Contain smaller DNA molecules a rod-shaped Bacillus called plasmids (provide extra functions, e.g. breaking down an a spiral form (Spirochete) antibiotic) Coccus (spherical shape) Bacillus (rod-shaped) Spirochete (spiral form) http://classes.midlandstech.edu/carterp/courses/bio225/chap04/lecture2.htm 16 Spherical (cocci) bacteria 1. Micrococci 2. Diplococci 3. Streptococci 4. Staphylococci 5. Tetrads 6. Sarcinae http://classes.midlandstech.edu/carterp/courses/bio225/chap04/lecture2.htm 17  Bacterial cell Site of protein Enable motility synthesis Serve an antiphagocytic function & used in production of vaccines Provide rigidity, strength & protection Enable bacteria to adhere to surfaces http://torresbioclan.pbworks.com/w/page/22377199/Prokaryotes%20and%20Eukaryotes 18 Bacterial cell  Cell wall confers rigidity upon bacteria and protects against osmotic damage  The rigid part of the cell wall is made of peptidoglycan  Gram's stain differentiates all bacteria into two distinct groups according to cell wall composition: Gram-positive organisms Gram-negative organisms  First described by Gram in 1884.  It is used to study morphologic appearance of bacteria. 19  Bacterial sporulation  Some bacteria (e.g. Bacillus and  Medical significance of Clostridium) can form spores sporulation: (endospores) Most notorious pathogens are enable them to survive adverse spore-formers including conditions, e.g. extreme Bacillus and Clostridium temperature, desiccation and Spores of these organisms can lack of nutrients remain viable for many years  Sporulation: process of endospore and are generally not killed by formation boiling  Spores germinate into new Spores can be killed by vegetative bacterial cells (cells autoclaving (i.e. subjecting the capable of growing and dividing) spores to temperatures above once condition becomes favorable. 120℃ at elevated pressure). 20  Generation Time (Doubling Bacterial Reproduction Time): time required for a cell to divide, e.g. E. coli - 20 minutes  Bacteria do not undergo the process of mitosis that our cells (and other eukaryotic cells) use.  They replicate their single Cell elongation circular chromosome to form two identical copies.  These copies are separated as the DNA replication cell grows, probably by attachment to the membrane.  Eventually, the cell pinches inward to separate the cell into Cross wall completed two smaller daughter cells, a process called binary fission.  By “Binary fission” - doubling the population (doubling time: 15-30 min-optimum) Daughter cells separate http://www.ncl.ac.uk/dental/oralbiol/oralenv/tutorials/bacterial_growth.htm 21 Bacterial Growth Curve 1. Lag phase - growth and reproduction are just beginning 2. Log phase - exponential phase (logarithmic increase in cell no.) 3. Stationary phase - cell growth (division) equals cell death 4. Death phase – cell deaths exceed cell growth (viable count 22 decline) Fungi  Eukaryotes  Multicellular: Molds and mushrooms  Have rigid cell walls (contain chitin)  Unicellular: Yeasts  Use organic chemicals for energy  Unique characteristics of molds in  Unlike plants, they are not their growth as networks of hyphae photosynthetic, but live by that form a mycelium breaking down organic materials.  Many fungi are decomposers but some cause plant and animal  Many produce elaborate fruiting diseases bodies during reproduction, such as mushrooms 23 http://www.microbeworld.org/what-is-a-microbe/microbe-gallery Molds  Multi- or unicellular  Grow in the form of mycelium  Reproduce by producing spores  Often associated with food spoilage (e.g. bread mold)  Some produce toxins, antibiotics and enzymes (useful in food production, e.g. cheese) http://classes.midlandstech.edu/carterp/Courses/bio225/chap12/lecture1.htm http://global.britannica.com/EBchecked/topic/78437/Rhizopus http://www.flora-balance.com/health_fungus.html 24  Yeast  Unicellular  Have an oval or spherical shape  Reproduce by budding  Budding: formation of a small cell (called a bud), and then pinches off the parent cell  Some are used for food production, e.g. alcohol fermentation, baking yeast in bread) Yeast in brewing Yeast leavener in baking 25 http://homecooking.about.com/od/specificfood/a/yeast.htm http://www.pha.jhu.edu/~ghzheng/old/webct/note7_3.htm http://blog.chicagometallicbakeware.com/2013/09/27/ask-experts-baking-yeast/ http://www.jimsbeerkit.co.uk/fermentation.htm Budding  Each bud forms at a new spot on the cell surface, and the bud gradually increases in size until it is as large as the parent cell, at which point the two cells separate. 26 Protists: Protozoa and algae Protozoa Algae  Eukaryotes  Eukaryotes  Absorb or ingest organic  Cell walls contain cellulose chemicals  Use photosynthesis for energy  May be motile via pseudopods,  Produce molecular oxygen and cilia, or flagella organic compounds Amoeba Green algae 27 http://www.nhm.ac.uk/research-curation/life-sciences/genomics-microbial- http://www.arcella.nl/pseudopodia diversity/organisms/algae/green-algae/index.html1  Viruses  Non-cellular (do not possess  Replicate only when they are in cellular organization) a living host cell  Contain RNA or DNA  Multiply by a complex process and not by binary fission  Contain a protein coat  Most viruses infect only specific  Some are enclosed by a lipid types of cells in one host envelope (enveloped or non- enveloped viruses)  They are unaffected by antibiotics  Some viruses have spikes  They are sensitive to Interferon 28 Hepatitis B virus HIV http://www.virology.net/Big_Virology/BVretro.html Influenza virus http://www.virology.net/Big_Virology/BVRNAortho.html http://www.virology.net/Big_Virology/BVDNAhepadna.html Morphology of viruses 29 Viral replication  Each virus tends to be specific for  Specificity is due to affinity of a certain kind of host. viral surface protein to proteins e.g. bacterial viruses (called on the surface of the host cells bacteriophages) cannot infect  Bacteriophages: have proteins animal cells, nor can animal in their tail fibers attracted to viruses infect bacterial cells. proteins on the surface of bacterial cells  Animal viruses: have protein spikes that are corresponding to proteins on the surface of animal cells 30 http://www.scienceprofonline.com/microbiology/what-is-a-virus.html Viral replication  Cellular stage of most virus infections follows similar processes: 1. The virus must attach to the (host) cell 5. The viral DNA or RNA is then copied surface. thousands of times, making the genetic material for new viruses. 2. The virus penetrates the plasma membrane and enters the cell. DNA viruses produce nucleotides and the DNA strands are then 3. The DNA or RNA of the virus sheds its directly copied from the original protective coating to give it access to the viral DNA. cell and reach the cell cytoplasm 6. The virus then uses the cell's ribosomes 4. The virus takes control of cell functions to create the proteins for the virus's to faciliate its reproduction. The virus‘s capsid genetic information begins to be read out by the cell (following the instructions of 7. The capsid begin to self-assemble, the viral genes rather than its own). The associating with the viral DNA or RNA normal cellular mechanisms are used for to complete viral particles. copying the viral DNA, making mRNA 8. The newly formed viruses (progency from viral DNA, and translating viral virus) are released from the cell into the mRNA into protein. outside environment. Viral replication: One-step growth curve In the growth of viruses, a single virus infects a cell and the infectious process produces many hundred or more new virus particles. A. Eclipse period: represents the time elapsed from initial entry and disassembly of the parental virus to the assembly of the first newly formed virus. During this period, active synthesis of virus components is occurring (most human viruses - 1 to 20 hrs) B. Exponential growth: the number of progeny virus produced within the infected cell increases exponentially for a period of time, then reaches a plateau 32 Sizes of viruses Copyright@2010 Pearson Education, Inc. 33 Measurement of microorganisms  Most microorganisms are in the micrometer (µm) size range  1 µm = 10-6 m = 10-3 mm  1 nm = 10-9 m = 10-6 mm  1000 nm = 1 µm 34  0.001 µm = 1 nm  Light Microscopy  Light microscopy: use visible light  In a compound microscope the to resolve objects image from the objective lens is magnified again by the ocular lens.  Visible light passes directly through the lenses and specimen  Total magnification = (called Bright-field microscopy) objective lens  ocular lens 35 http://www.freeinfosociety.com/media/images/1250.jpg  Characteristics of microscopes Type Characteristics Resolving power Light Observe morphology of microorganisms such 0.2 µm microscope as bacteria, protozoa, fungi and algae in living (unstained) or non-living (stained) state Cannot observe microbes less than 0.2 µm (e.g. viruses) Electron TEM (Transmission electron microscope) 0.2 nm microscope Examine cellular and viral ultrastructure Specimen is non-living SEM (Scanning electron microscope) 20 nm Examine surface features of cells and viruses Gives three-dimensions Specimen is non-living http://qph.cf.quoracdn.net/main-qimg-d1f3c489a39f538ee71105c8cb756bc3 http://media-cache-ak0.pinimg.com/736x/6f/7a/1d/6f7a1d993b6afa272d36ede1eea0d23e.jpg 36 http://ucsdnews.ucsd.edu/news_uploads/hiv-particles-leaving-cell-deerinck.jpg  Gram staining  Gram staining: based on different composition in cell wall of bacteria  Gram-positive bacteria: thick cell wall and no outer membrane → blue-purple colour  Gram-negative bacteria: thin cell wall and an outer membrane → pink-red colour Gram + Gram - Peptidoglycan Peptidoglycan blue-purple pink Lipopolysaccharide & protein (outer membrane) http://biocene.blogspot.com http://medicinexplained.blogspot.hk/2012/02/gram-staining-procedure-mechanism.html http://water.me.vccs.edu/courses/env108/changes/gram.jpg 37 Difference between Gram positive and Gram negative bacteria http://www.zo.utexas.edu/faculty/sjasper/images/27.5.jpg 38  Gram staining Color of Color of Gram + cells Gram – cells Primary stain: Purple Purple Crystal violet Mordant: Iodine Purple Purple Decolorizing agent: Purple Colorless Alcohol-acetone Counterstain: Safranin Purple Pink http://medicinexplained.blogspot.hk/2012/02/gram-staining-procedure-mechanism.html 39 Gram staining (I) Smear preparation (II) Staining procedures  Fixation: 1) Staining with crystal violet.  Smear the bacterial sample on 2) Fixation with iodine stabilizes the slide (use aseptic crystal violet staining. All bacteria technique) and left to air dry remain purple or blue.  Heat fixing the smear by 3) Decolorization with alcohol or carefully passing the slide other solvent. Decolorizes some through the Bunsen burner bacteria (Gram negative) and not (avoid burning the sample) others (Gram positive). 4) Counterstaining with safranin. Gram positive bacteria are already stained with crystal violet and remain purple. Gram negative bacteria are stained pink. http://pathmicro.med.sc.edu/fox/gram-st.jpg  Gram stain showing bacterial population (gram-positive cocci) on the smear. (4+: >30/HPF, 100x objective magnification)  Diagnostic isolation and identification of bacteria  Basic laboratory techniques: Direct microscopic visualization of the organism, Cultivation and identification of the organism, Detection of microbial antigens, Detection of microbial DNA or RNA (nucleic acid) Detection of microbial protein pattern 42 Copyright@2013 Wolters Kluwer Health| Lippincott Williams & Wilkins Part III: Microbes and You 43  Where can we find microorganisms? Microbes are everywhere.  In environment: Water Soil Air Foods http://www.cdc.gov/leprosy/transmission/index.ht http://ecofriend.com/wp-content/uploads/2012/07/water_contamination.jpg http://www.azphm.com/images/EarthTalkChlorineTapWater.jpg 44 http://www.technology.org/2014/05/05/lactobacilli-milk-oral-cavity-may-help-fend-deadly-p-aeruginosa-bacteria/ http://www.ourfood.com/Moulds_Yeasts.html http://endtimeheadlines.wordpress.com/2013/07/26/as-the-government-continues-to-shut-down-family-farms-your-ability-to-make-good-food-choices-diminishes/  Where can we find microorganisms? Microbes are everywhere.  In human body (e.g. on the skin/ in our intestines): Micrococcus – commonly isolated from the E. coli - In our intestine skin and nasal membranes of humans 45 http://topnews.us/content/213325-bacteria-hands-can-reveal-your-identity http://www.permaculturenews.org/images/gut-microbiome.jpg http://textbookofbacteriology.net/Micrococcus.jpeg http://www.knowabouthealth.com/wp-content/uploads/2011/10/e-coli.jpg Are microbes beneficial or harmful? Microbes can be:  Normal microbiota (non-pathogenic, does not cause disease)  Pathogens (cause disease in human)  Opportunistic pathogens (do not cause disease under ordinary conditions but await the opportunity to cause infections) 46 Normal microbiota  Non-pathogenic micro-organisms that live in or on a human body without causing disease  Live in symbiosis with the human body  Usually found in areas of the body exposed to external environment (e.g. skin, nose, mouth, intestinal tract, urogenital tract)  Usually helpful to humans and their environment  Very abundant: a normal human has approximately 1013 body cells and 1014 individual normal microbiota  However, microorganisms also tend to be very small, bacteria especially are much smaller than are our own cells 47 Where are normal microbiota in human?  Nose and mouth  Skin  Respiratory system  Digestive system (Gastrointestinal tract)  Genitourinary system (Urinary and reproductive systems) 48 http://www.scq.ubc.ca/microbes-and-you-normal-flora/ Symbiotic microbe-host relationships Symbiosis (Symbiotic relationships): living together or close association of two different organisms (host = human) Types Microbe-host relationships Examples Mutualism both benefit E. coli bacteria in the large intestine Commensalism one organism benefits Staphylococcus and the other is epidermidis on the unaffected – no benefit/ skin harm Parasitism one benefits and the H1N1 virus particles other is harmed on a host cell 49 Mutualism What are the benefits?  Benefits to the human  Benefit to the bacteria Bacteria may produce Have a place to survive and vitamins (e.g. vit. B and K) multiply Break down food that host Obtain food source can’t normally digest. Protect host - Microbial antagonism (suppress the growth of pathogenic organisms): Compete for nutrients Produce inhibitory substances 50 Pathogens  Pathogen has the ability to cause  Opportunistic pathogens are disease in a susceptible host normal microbiota (human) They do not cause disease under  Pathogens cause TWO major types normal conditions but cause of diseases: disease under special conditions Infectious diseases (pathogen e.g. E. coli, commonly found in cause disease) – when a the intestine, can cause urinary pathogen colonizes the body tract infections if introduced and subsequently cause disease into the bladder. Microbial intoxication (toxin Immunosuppression can allow cause disease) – when a person otherwise harmless bacteria to ingests a toxin (poisonous cause disease. substance) produced by a AIDS, some cancer treatments pathogen in vitro (outside the and transplant rejection drugs body) all suppress the immune system and allow the normal microbiota to cause occasionally serious disease. 51 Part IV: Pioneers of Microbiology 52 Pioneers of Microbiology  Robert Koch: proved that a microbe caused disease Koch’s Postulates: specific microbe cause a specific disease  Louis Pasteur: disproved spontaneous generation Demonstrated that life did not arise spontaneously from nonliving matter Pasteurization: kill pathogens in many types of liquids Fermentation  Alexander Fleming: observed Penicillium fungus inhibited the growth of bacterial culture Penicillin used clinically as antibiotic (substances produced naturally by bacteria and fungi to inhibit the growth of other microorganisms 53 Koch’s Postulates  Anthrax 1. Observe the microorganism in the sick host. 2. Grow the microorganism in the pure culture. 3. Inoculate the microorganism into a healthy, susceptible host-and cause symptoms in that host. 4. Isolate the microorganism in the pure culture. 54 Koch’s Postulates Robert Koch was the first Koch’s Postulates defines: to prove that a microbe The suspected pathogenic caused disease. organism should be present in In the blood of animals all cases of the disease and dying of anthrax there was absent from healthy animals always one type of bacterium: The suspected organism Bacillus anthracis should be grown in pure culture Cells from a pure culture of the suspected organism should cause disease in a healthy animal The organism should be re- isolated and shown to be the same as the original. 55  Pasteur's test of spontaneous generation  Louis Pasteur's experiment that disproved spontaneous generation 56 http://202.204.115.67/jpkch/jpkch/2008/wswx/chapter%201.htm Alexander Fleming  He observed bacterial Staphylococci colonies disappearing on plates contaminated with mold. Fleming extracted the compound from the mold responsible for destruction of the bacterial colonies. The product of the mold was named penicillin, after the Penicillium mold from which it was derived. Discovery of antibiotics Penicillium mold inhibit the growth of bacteria 57 http://classes.midlandstech.edu/carterp/Courses/bio225/chap01/sld005.htm Exercise 1