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Medical Microbiology Introduction: Cell Structure & Microorganisms
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Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level ILOs • Define microbiology and microorganisms. • Describe the system of scientific nomenclature of microorganisms. • Identify main types of microorganisms • Differentiate between eukaryotic and prokaryotic cell. 2020/2021 Maha Fathy 2 What is Microbiology • Micro= tiny (very small) • Bio= living • Logy= the study of • The study of very small (tiny) living organisms = The study of microorganisms 2020/2021 Maha Fathy 3 Microorganisms • They are very small living organisms including bacteria, fungi, viruses, protozoa and helminths. • Most of them cannot be seen by naked eye • They need microscopes to be seen • Bacteria • Fungi Ordinary light microscope • Viruses Electronic microscope 2020/2021 Maha Fathy 4 Role of microorganisms in health and disease • A minority of microorganisms are pathogenic (capable to produce disease) • Many of them are colonizing human body as normal microbiota • The majority of them play a vital role in supporting and maintaining nature and life (saprophytic). 2020/2021 Maha Fathy 5 Nomenclature of Microorganisms • In bacteria & fungi: Genus Starts with capital letter 2 words Staphylococcus aureus Starts with small letter Candida albicans In viruses: single (poliovirus) or two names (not genus and species (herpes simplex) usually describe clinical condition. 2020/2021 Maha Fathy 6 Types of Microorganisms Microorganisms Cellular Prokaryotic 2020/2021 Non cellular Eukaryotic Maha Fathy Viruses 7 Prokaryotic Cell = primitive cell (Bacterial cell) • Smaller (0.1-5 µm) • NO true nucleus (single DNA molecule, located inside cytoplasm, NO histones) • NO membrane bound intracytoplasmic organelles: • No mitochondria (respiratory enzymes ae located in cell membrane) • No Endoplasmic reticulum • Smaller ribosomes (70 S; 2 subunits: 50 S & 30 S) • Cell wall is present (except Mycoplasma species): peptidoglycan in bacteria, chitin in fungi • No sterol in cell membrane ( lipoprotein only) (except Mycoplasma species) • Division: Simple binary fission 2020/2021 Maha Fathy 8 Eukaryotic Cell = True cell (Fungal or Human cell) • Size: Larger (10-100 µm) • True nucleus (nuclear membrane): present • Chromosomes: multiple, DNA is associated with histones • Membrane bound organelles (Mitochondria & End. Reticulum): present • Site of respiratory enzymes: mitochondria • Ribosomes: larger (80 S; 60 S & 40 S) • NO Peptidoglycan Cell Wall • Human cell: no cell wall • Fungal cell: chitin cell wall • Sterol in cytoplasmic membrane (lipoprotein+ sterol) • Mitotic division 2020/2021 Maha Fathy 9 1. Nucleoid, Circular simple DNA 1. True nucleus, Linear complex DNA DNA 2. No sterol in cell membrane Cell membrane 2. Sterol in cell membrane 3. No membrane bound intracytoplasmic structures Cytoplasm 3. Membrane bound intracytoplasmic structures 4. Smaller ribosomes 2020/2021 Ribosomes Maha Fathy 4. Larger ribosomes 10 Cell wall is a characteristic for prokaryotes Extra cellular structures Cell Wall Peptidoglycan containing cell wall in bacteria 9/22/2019 Maha Fathy 11 Compare between prokaryotic & eukaryotic cells giving an example for each. Give reasons Bacterial cell is prokaryotic in nature (not a true cell) What is the most important feature making scientists identifies a newly discovered microorganism as a bacterium? 2020/2021 Maha Fathy 12 Most welcome for questions 2020/2021 Maha Fathy 13 Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level ILOs 1. Demonstrate different components of bacterial cell. 2. Compare and contrast cell wall of Gram-positive & Gram-negative Bacteria. 3. Differentiate protoplast, spheroplast, L form, and mycoplasma. 4. Describe the structure and functions of bacterial plasma membrane. 5. Differentiate the functions and structure of the nucleoid, ribosomes and inclusions. 6. Describe the structure and function of the capsule. 7. Compare structure and functions of flagella and pili. 8. Describe the functions of spores, sporulation, and spore germination. 2020/2021 Maha Fathy 2 ILOs 1. Demonstrate different components of bacterial cell. 2. Compare and contrast cell wall of Gram-positive & Gram-negative Bacteria. 3. Differentiate protoplast, spheroplast, L form, and mycoplasma. 4. Describe the structure and functions of bacterial plasma membrane. 5. Differentiate the functions and structure of the nucleoid, ribosomes and inclusions. 6. Describe the structure and function of the capsule. 7. Compare structure and functions of flagella and pili. 8. Describe the functions of spores, sporulation, and spore germination. 2020/2021 Maha Fathy 4 Bacterial Cell Structure Extra cellular structures Cell Wall Cytoplasm + Intra-cytoplasmic structures Cell (cytoplasmic) membrane 2020/2021 Maha Fathy 5 2020/2021 Maha Fathy 6 Cell wall It gives the bacteria its shape (rigid structure) Cocci Bacilli Curved Bacilli Cocco Bacilli Spirochetes 3/18/2021 Maha Fathy 7 Reaction of bacteria to Gram stain Gram Positive Gram positive bacilli 2020/2021 Gram Negative Gram negative bacilli Gram negative cocci Gram positive cocci Maha Fathy 8 Cell Wall structure in gram positive & gram negative bacteria 2020/2021 Maha Fathy 9 Peptidoglycan Structure (murein or mucopeptide) 2020/2021 Maha Fathy 10 Cell Wall of gram-positive bacteria Thick peptidoglycan layer (repeating sugar subunits linked by peptide side chains) Teichoic acid & lipoteichoic acid (Polymers of glycerol or ribitol phosphate, major surface Ag) 2020/2021 Maha Fathy 11 Cell Wall of gramnegative bacteria Thin peptidoglycan layer Outer membrane formed of Lipopolysaccharides (Endotoxins) Peri-plasmic space Outer membrane proteins & porins 2020/2021 Maha Fathy 12 Lipopolysaccharides of gram negative bacteria 2020/2021 Maha Fathy 13 Test your self What are the unique structures? Gram Positive Bacteria Gram Negative Bacteria ?????? ?????? 2020/2021 Maha Fathy 14 Cell Wall Function 1. 2. 3. 4. 5. 6. 7. Maintains the shape of the bacterial cell (cocci, bacilli … etc). Supports the weak cytoplasmic membrane. Provide osmotic protection Plays a role in cell division. Responsible for the antigenicity (O Ag in gram –ve bacteria & Teichoic acid in gram +ve bacteria) It contains associated proteins: porins for selective permeability & adhesins for attachment Responsible for staining properties of the organism. 2020/2021 Maha Fathy 15 Functions of Outer Membrane (of gram-negative bacteria) • It protects the peptidoglycan from effects of lysozyme • It impedes the ingress of many antibiotics • It is associated with proteins: • Porins: allow diffusion of some material • Adhesins: attachment 2020/2021 Maha Fathy 16 Click to edit Master title style • Edit Master text styles • Second level CELL WALL DEFICIENT BACTERIA • Third level • Fourth level • Fifth level 2020/2021 Maha Fathy 17 Cell wall deficient bacteria Naturally occurring • Mycoplasma • Sterol containing Spontaneously (or artificially) induced) • L-Forms plasma Lab Induced • Protoplast • Spheroplast membrane 2020/2021 Maha Fathy 18 Cell wall deficient Bacteria Naturally occurring: Mycoplasma • Smallest type of bacteria • No defined cell shape • Resistant to antibiotics which act on cell wall • Resistant to lysozyme 2020/2021 Maha Fathy 19 Lysozyme breaks this bond 2020/2021 Maha Fathy 20 L-Forms • Can occur Spontaneously (or induced) from some bacterial genera ( Bacillus, Clostridium, Haemophilus, Pseudomonas, Staphylococcus, and Vibrio). By action of antibiotics which can inhibit cell wall synthesis NB: First discovered in Lister Institute in London 2020/2021 Maha Fathy 21 Medical importance of L-forms • L-forms can remain viable inside the host • They can replicate • They are not killed by antibiotics acting on cell wall • L-forms can revert to the normal form on removal of the inhibitor producing relapses of infection. 2020/2021 Maha Fathy 22 Lab induced cell wall deficient bacteria Protoplast • Originate from Gram positive bacteria Spheroplast • Originate form Gram negative bacteria Cell membrane 2020/2021 Maha Fathy Outer membrane 23 Spheroplast Protoplast • From gram positive bacteria • From Gram negative bacteria • Induced by lysozyme • Induced by action of lysozyme on EDTA-treated gram negative cell (or action of antibiotics) • All cell wall material has been removed • Spherical structure surrounded by one membrane 2020/2021 • Still retaining part of cell wall (outer membrane) • Spherical structure surrounded by two membranes Maha Fathy 24 How are these cell wall deficient bacteria kept without rupture??? • By being maintained in an osmotically balanced medium • (A solution that has the same concentration of ions and small molecules as cytoplasm) 2020/2021 Maha Fathy 25 Test your self •What is the medical importance of these altered forms of bacteria with weakened cell wall? 2020/2021 Maha Fathy 26 • We want to synthesize an antibacterial agent (antibiotic) to be highly selective (acting exclusively on bacterial cell and not affecting human host cells). • What is the ideal target of this antibiotic??? 2020/2021 Maha Fathy 27 Most welcome for questions 2020/2021 Maha Fathy 28 Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level ILOs 1. Demonstrate different components of bacterial cell. 2. Compare and contrast cell wall of Gram-positive & Gram-negative Bacteria. 3. Differentiate protoplast, spheroplast, L form, and mycoplasma. 4. Describe the structure and functions of bacterial plasma membrane. 5. Differentiate the functions and structure of the nucleoid, ribosomes and inclusions. 6. Describe the structure and function of the capsule. 7. Compare structure and functions of flagella and pili. 8. Describe the functions of spores, sporulation, and spore germination. 2020/2021 Maha Fathy 2 STRUCTURES INTERNAL TO CELL WALL Cytoplasmic membrane & mesosomes 2020/2021 Cytoplasm & Intra cytoplasmic structures Maha Fathy 3 Cytoplasmic Membrane Structure • Composed of protein and phospholipids • In contrast to eukaryotic cells: • it is rich in protein with little phospholipids (no sterol except in mycoplasma) 2020/2021 Function • Active transport and selective permeability • Involved in protein secretion (extracellular enzymes and exotoxins) (No endoplasmic reticulum in prokaryotic cell) • It contains cytochrome enzyme responsible for respiration and energy production (No mitochondria in prokaryotic cell) • Plays an important role in cell division Maha Fathy 4 Mesosomes • They are invaginations of the cytoplasmic membrane inside the cytoplasm. • Two types of mesosomes are known as lateral and septal. • Septal mesosomes are involved in cell division where bacterial DNA is attached. • They increase the surface area of the cytoplasmic membrane thus increasing the efficiency of permeability and active transport. • It is the site for respiratory enzymes. 2020/2021 Maha Fathy 5 Role of septal mesosomes in cell division 2020/2021 Maha Fathy 6 Intra Cytoplasmic Structures 2020/2021 Maha Fathy 7 Intracellular Structures •Genetic material •Ribosomes: protein synthesis •Storage granules or inclusion granules 2020/2021 Maha Fathy 8 Genetic material •Bacterial chromosome •Extra chromosomal DNA: plasmid 2020/2021 Maha Fathy 9 Bacterial Chromosome • Single molecule of double stranded DNA (double helix) • Each strand is a long sequence of nucleotide units • The bacterial chromosome carries the essential genetic materials (determinants) that control the bacterial properties and behavior Sugar Phosphate backbone Hydrogen bonds Base Pairs • It is a circular molecule free inside cytoplasm 2020/2021 Maha Fathy 10 2020/2021 Maha Fathy 11 2020/2021 Maha Fathy 12 The 2 strands are complementary Replicates by semiconservative method 2020/2021 Maha Fathy 13 Plasmid • Extra chromosomal double stranded circular DNA. • Carry certain genetic information, e.g. antibiotic resistance, toxin production Genetic information carried by plasmid are not essential for bacterial life 2020/2021 Maha Fathy 14 Bacterial Plasmid: Define? • Extrachromosomal DNA • They are dispensable (not necessary for life of the cell) • They are capable of self replication (independent of cell replication) (autonomous) • Many copies of the same plasmids may present in the same cell. • Different plasmids may coexist within the same bacterial cell. • Plasmids can be transferred from one bacterium to another. • Plasmids can be integrated into the bacterial chromosome (Recombination). • Plasmids occur in both Gram-positive and Gram-negative bacteria. 2020/2021 Maha Fathy 15 Ribosomes • The site of protein synthesis. • They have sedimentation coefficient of 70S being composed of 2 subunits: small 30S and large 50S subunits. • Aggregates of multiple ribosomes on a m-RNA are called polysomes Human cell ribosomes are 80S; formed of 2 subunits: 60S & 40S 2020/2021 Maha Fathy 16 Inclusion granules • Round granules observed in cytoplasm of many bacteria. • These are not permanent or essential structures. • They are either stored energy or nutrient reserve concerned with cell metabolism, • e.g. volutin granules (metachromatic granules of Corynebacterium diphtheriae). 2020/2021 Maha Fathy 17 STRUCTURES EXTERNAL TO CELL WALL 2020/2021 Maha Fathy 18 Capsule • Found in some bacteria • Mostly formed of high molecular weight polysaccharides (antigenic) or poly peptide • Can not be seen in Gram stain preparations (special stain) • An important virulence factor • Capsular antigens are used for diagnosis & Typing 2020/2021 Maha Fathy 19 The capsule as an important virulence factor • Antiphagocytic • Help in attachment of bacteria to mucous membranes • Formed only in vivo 2020/2021 Maha Fathy 20 Test your self Give reason Capsular polysaccharides are used for preparation of vaccines against some bacteria. 2020/2021 Maha Fathy 21 Slime (Glycocalyx) • Excreted by some bacteria as loosely attached polysaccharide material • Plays an important role in attachment of bacteria to surfaces and formation of biofilms on biotic or abiotic surfaces • It plays an important role in pathogenesis of many diseases (as cystic fibrosis and dental caries) • Can lead to healthcare associated infection when attached to devices inserted in human host as IVDs or urinary catheters or prosthetics like artificial valves or artificial joints 2020/2021 Maha Fathy 22 Flagella & Fimbria (Pili) 2020/2021 Maha Fathy 23 Flagella • Formed of proteins (Flagellin) • Found in some gram negative bacteria • Make bacteria motile (organelle of motility) • An important virulence factor • They are antigenic and can stimulate anti-falgellar antibodies 2020/2021 Fimbria (Pili) • Formed of glycoproteinprotein (Pilin) • Found mainly in gram negative bacteria • They are important virulence factors enabling bacteria to adhere to mammalian cells (colonization) • Specialized pili are involved in gene transfer (sex pilus) in a process called conjugation Maha Fathy 24 Polar/Monotrichous Lophotrichous Amphitrichous Peritrichous 2020/2021 Maha Fathy 25 Test your self Give reason Flagella & pili can play an important role in pathogenicity of some bacteria Mention two functions of bacterial fimbria (pili) 2020/2021 Maha Fathy 26 BACTERIAL SPORES 2020/2021 Maha Fathy 27 Spores • Metabolically dormant forms of some species (Bacillus & Clostridia) • Spore formation occurs outside the body in response to unfavorable conditions, e.g., depletion of nutrition, accumulation of metabolites or unsuitable gaseous environment • They are highly resistant to dryness, heat and chemicals as disinfectants • They can live in soil for many years 2020/2021 Maha Fathy 28 Spores (cont.) • Spores are formed outside the body, • Can not be stained by ordinary stains like gram stain (need special spore stain). • In germination each spore gives rise to a single vegetative cell which can multiply (it is not a way of reproduction). 2020/2021 Maha Fathy 29 Sporulation process • The plasma membrane invaginates enclosing section of cytoplasm that contain bacterial chromosome, some ribosomes, and other cytoplasmic materials. • It acquires thick covering protective keratin-like coat that is responsible for the remarkable resistance of the spore to heat, dehydration, radiation, and chemicals. 2020/2021 Maha Fathy Septum from invagination of cell membrane Fore spore Endospore maturation Death of mother cell and release of endospore 30 2020/2021 Maha Fathy 31 Resistance of bacterial spores Marked resistance of endospores is attributed to: • Thick outer keratin like coats (high resistance to heat & chemicals) • High content of calcium dipicolinate (protects bacterial DNA) • Low water content • Very low metabolic and enzymatic activity 2020/2021 Maha Fathy 32 Test your self • Which of the following can be used ideally as an indicator of sterilization process? • Vegetative bacteria • Bacterial spores 2020/2021 Maha Fathy 33 Test your self Give reasons: • In contrast to fungi, bacterial spore formation is not considered a method of reproduction. • Bacterial spores are formed outside the body of the host while capsules are formed inside host’s body 2020/2021 Maha Fathy 34 Test your self • Compare between the cell wall structure in gram-positive and gram-negative bacteria • Mention the structures present in gram positive but not in gram negative bacteria • Mention the structures present in gram negative but not in gram positive bacteria • What are the components of the bacterial cell structure essential for life? 2020/2021 Maha Fathy 35 Most welcome for questions 2020/2021 Maha Fathy 36 Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level ILOs 1. Define bacterial growth, and generation time. 2. Describe the process of bacterial replication. 3. Demonstrate the phases of microbial growth and describe their relation to generation time. 4. Discuss clinical significance of growth curve. 5. Compare and contrast between autotrophic and heterotrophic bacteria regarding nutritional requirements. 6. Differentiate bacteria according to their gaseous requirements. 7. Describe physical conditions necessary for bacterial growth. 2020/2021 Maha Fathy 2 Bacterial Growth • The accumulation of biomass and genomic replication, cell division and an increase in the number of cells • It occurs by simple binary fission; each bacterial cell forms two equal-sized daughter cells. 2020/2021 Maha Fathy 3 Bacterial Multiplication: Simple Binary Fission • The cell became elongated • Chromosomal replication takes place: where the double strand DNA separates, each strand acts as a template for synthesis of new strand by DNA polymerase enzyme, to produce two identical “daughter” copies • Cell wall and cytoplasmic membrane begin to constrict • Cross-wall forms separating the 2 daughter cells 2020/2021 Maha Fathy 4 Rate of Bacterial Growth • Time required for bacteria to double their number in culture media is called the generation time • Some bacteria have short generation time: (20 minutes for E.coli, 13 minutes for Vibrio cholerae) • Some bacteria have long generation time e.g. Mycobacteria (18 hours) 2020/2021 Maha Fathy 5 Bacterial Growth in vitro • Most of bacteria can grow on artificial culture media (fluid or solid media) • Some bacteria are obligate intracellular (Chlamydia & Rickettsia): grow on tissue culture • Treponema pallidum (of syphilis) and Mycobacterium leprae (of leprosy) can not grow in vitro 2020/2021 Maha Fathy 6 Forms of in vitro bacterial growth Fluid Media Solid Media • Bacterial Growth is indicated by turbidity 2020/2021 • A single bacterium supplied with the appropriate growth requirements will multiply on the solid medium to from a colony Maha Fathy 7 Click to edit Master title style • Edit Master text styles Bacterial Growth Dynamics (Bacterial Growth Curve) • Second level • Third level • Fourth level • Fifth level 2020/2021 Maha Fathy 8 Bacterial Growth Curve 2020/2021 Maha Fathy 9 Lag phase • No increase in the number of cells • They adapt to their new environment. • They start to synthesize enzymes and molecules required for bacteria growth (they may increase in size) • Its length depends on: • type of the organism, • size of the inoculum • type of growth medium 2020/2021 Maha Fathy 10 Log (exponential) Growth phase • It is the most active phase of bacterial growth. • The number of bacteria increases steadily by time. • This phase continues until the nutrient of the medium is exhausted or toxic metabolites accumulate. Bacteria are highly susceptible to antibiotics at this phase. 2020/2021 Maha Fathy 11 Stationary phase • Exhaustion of nutrients and accumulation of toxic products • Decrease in bacterial growth • Bacterial death is balanced by formation of new cells • The number of viable bacteria remains constant 2020/2021 Maha Fathy 12 Decline phase • The death rate increases and exceeds the multiplication rate. • This is due to nutrient exhaustion and accumulation of toxic products 2020/2021 Maha Fathy 13 Applications on bacterial growth curve 1 2020/2021 2 3 3 Stages of infectious disease 1. Incubation Period 2. Clinical disease (signs & symptoms) 3. Recovery & Convalescence Maha Fathy 14 • In which phase of the bacterial growth curve do sporulation of spore forming bacteria start to occur? 2020/2021 Maha Fathy 15 Requirements of Bacterial Growth 2020/2021 Maha Fathy 16 Requirements for Bacterial Growth Chemical: • Nutritional requirements: Carbon & Nitrogen sources and mineral elements • Gaseous requirements (Oxygen & CO2) Physical • Temperature • Hydrogen ion concentration (pH) • Osmotic pressure 2020/2021 Maha Fathy 17 Carbon & Nitrogen Sources • Medically important bacteria require organic compounds as their carbon sources and called Heterotrophes • Autotrophic bacteria synthesize organic metabolites from CO2, water and mineral salts (saprophytic, free living bacteria) . 2020/2021 Maha Fathy 18 Heterotrophic bacteria Exacting (fastidious): require both organic form of carbon and nitrogen for growth. • They have complicated nutritional requirements; • Grow only in special artificial cultures • Usually they need specific growth factors including yeast extract, blood, B complex vitamins, amino acids, purines and pyrimidines Non-Exacting: Can assimilate organic source of carbon and inorganic source of nitrogen. 2020/2021 Maha Fathy 19 Oxygen Requirements • Facultative anaerobe: E.coli • Obligate anaerobe: Clostridia • Obligate aerobe: Pseudomonas • Microaerophilic: Campylobacter • Aerotolerant (obligate fermenters): lactobacillus 2020/2021 Maha Fathy 20 Obligate aerobe • They have absolute requirement for oxygen • They use O2 as the final electron acceptor in the process of oxidative phosphorylation (aerobic respiration) 2020/2021 Maha Fathy 21 • In aerobic respiration the final electron acceptor is O2 to give free radicals as superoxide anions (O2-) and hydroxyl radicals (OH-) • These products are toxic to the cell • They should be eliminated by certain enzymes 2O2- + 2 H 2H2O2 2020/2021 Superoxide dismutase O2 + H2O2 2 H2O + 2 O2 Catalase Maha Fathy 22 Obligate anaerobe • They are killed by oxygen (why?) • They produce energy through anaerobic respiration (final electron acceptor is inorganic substance other than oxygen) or fermentation (substrate phosphorylation) 2020/2021 Maha Fathy 23 Facultative anaerobe • They are able to grow in either the presence or the absence of oxygen. However, better growth occurs in the presence of O2 2020/2021 Maha Fathy 24 • Microaerophilic: require limited amounts of oxygen (5%) for aerobic respiration • Aerotolerant: Not killed by oxygen but not multiply in the presence of it (obligate fermenters) 2020/2021 Maha Fathy 25 Carbon Dioxide • Bacteria need CO2 for growth • Most of bacteria need CO2 in air concentration • Few bacteria require additional CO2 (5-10%) for growth (e.g. pathogenic Neisseria) 2020/2021 Maha Fathy 26 Temperature • Most pathogenic bacteria grow optimally at 37oC • Optimum temperature for few bacteria may be higher e.g. 42oC for Campylobacter spp. • Some bacteria can grow on low temperature (0-4oC) e.g. Lysteria monocytogenes ( important in food borne illness) 2020/2021 Maha Fathy 27 Bacterial temperature range •Psychrophilic: range of (5 – 25oC) •Mesophilic: range of (25 – 45oC) • Thermophilic: range of (45 – 75oC) 2020/2021 Maha Fathy 28 Hydrogen ion Concentration: ( pH) • pH refers to the acidity or alkalinity of a solution. • Most pathogenic bacteria grow best at pH in the range of 7.2-7.6 (near neutrality) • Few exceptions: • Lactobacilli in the vagina prefers acidic pH (e.g. 4) • Vibrio cholerae prefers alkaline pH (e.g. 8) 2020/2021 Maha Fathy 29 Osmotic pressure • Most microorganisms grow in optimum osmotic pressure (the salt concentration of microbial cytoplasm is the same as the external environment ; 1% NaCl; isotonic). • Halophilic bacteria: live in marine environments and can tolerate high salt concentrations. 2020/2021 Maha Fathy 30 What is the main event characterizing the phase (A) in the corresponding figure of bacterial growth curve? • Accumulation of metabolic intermediates • Adaptation of the bacteria to the new environment • Depletion of nutrients • Germination of spore forming bacteria • Increased susceptibility to antibiotics 2020/2021 Maha Fathy 31 Most welcome for questions 2020/2021 Maha Fathy 32 Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level ILOs • Discuss relationship between microbes and their hosts. • Explain importance of normal flora in health and disease. • Differentiate between colonization and infection. • List factors affecting host microbe relationship. • Discuss different bacterial virulence factors. • Compare between endotoxin and exotoxins. 3/28/2021 Maha Fathy 2 Host Microbe Relationships • Parasitic (pathogenic) • Pathogenic Bacteria • Commensal • Normal Flora • Mutual • Vit K producing gut flora 3/28/2021 Maha Fathy 3 What is normal flora? Conjunctiva Oral cavity URT Skin GIT Urogenital tract 3/28/2021 Maha Fathy 4 How is it acquired? 3/28/2021 Maha Fathy 5 What are their types? Resident Transient Always present on /in the human body 3/28/2021 Temporarily present under certain condition Maha Fathy 6 What are the benefits of normal flora? Biological Barriers • Prevent colonization of pathogens through competition • Cover the binding sites (receptors) used for attachment • Consume available nutrients • Produce components that are toxic to other bacteria (Bacteriocins) 3/28/2021 Maha Fathy 7 • Bacteria can provide some nutrients e.g. Vitamin K, and also aid in digestion, and absorption of nutrients. • Bacterial colonization of the newborn infants acts as a powerful stimulus for the development of immune system 3/28/2021 Maha Fathy 8 What are the drawbacks of normal Flora? • Endogenous source for infection Opportunistic pathogens 3/28/2021 Maha Fathy 9 Principles of Infectious Diseases 9/22/2019 Maha Fathy 10 Possible outcomes following exposure to microorganisms Exposure Elimination Colonization The microbe is eliminated by host defenses without affecting the host. The microbe has become established and is multiplying in / on body surface of a host. 9/22/2019 Maha Fathy 11 Possible outcomes following Colonization Colonization Part of normal flora • Transient • Resident 9/22/2019 Infection • Unapparent: subclinical infection (no symptoms & signs) • Clinical Infection (infectious disease) Maha Fathy 12 Principles of Infectious Diseases • Exposure: source of microorganisms • Colonization: microbe has become established and is multiplying • Infection: detrimental effect on the host (unapparent or latent) • Infectious Disease: clinical infection; symptoms & signs 3/28/2021 Maha Fathy 13 Important Definitions • Pathogenicity The ability of the microorganism to cause disease. • Virulence The degree of pathogenicity of an organism • Virulence factors Factors help the microbe to cause infection and disease 3/28/2021 Maha Fathy 14 Types of Pathogens • Primary Pathogens: e.g., diphtheria bacilli • cause disease in healthy individuals • Opportunistic Pathogens: e.g., uropathogenic E. coli & coagulase negative staphylococci • Normal flora when introduced into unusual location • In immunocompromised host 3/28/2021 Maha Fathy 15 Differentiate?? • Clinical infection & Subclinical infection • Case & Carrier 3/28/2021 Maha Fathy 16 Factors Affecting the Host-Microbe Relationship Microbial factors • Virulence factors Host factors • Immune response: • Innate • Acquired 3/28/2021 Maha Fathy 17 Bacterial Virulence Factors A. The ability to adhere to host cells and resist physical removal B. The ability to resist immediate destruction by host immune defense C. Obtaining required iron D. Invasion and intra cellular survival E. Type III secretion system dependence virulence in gramnegative bacteria F. Damage to host cells by inflammatory response or bacterial products 3/28/2021 Maha Fathy 18 A. The ability to adhere to host cells and resist physical removal Fimbriae (Pili) • Uropathogenic E. coli Non fimbrial adhesins • E.coli : Diarrhogenic • Moraxella, Hemophilus, Bordetella • Neisseria • Receptors: • Urinary tract epithelium • RBCs 3/28/2021 Maha Fathy 19 Other bacterial structures help in adherence • Teichoic acid in gram positive bacteria • Capsule (Pneumococci) • Glycocalyx (biofilm formation) e.g., Coagulase negative Staphylococci Bacterial biofilm 9/22/2019 Maha Fathy 20 B. The ability to resist host immune defense • Capsule (Pneumococci) • Immunoglobulin A protease (Pneumococci) • Cell wall proteins: bind to Fc portion of Ab preventing its action. • Protein A (Staphylococcus aureus) • Protein M (Streptococcus pyogenes) • Intracellular survival (Mycobacterium tuberculosis, Listeria monocytogenes) 3/28/2021 Maha Fathy 21 C. Obtaining required iron: The ability to compete for iron • Iron is essential for bacterial growth • Siderophores: bacterial iron chelators • Siderophores are synthesized and excreted by the bacterium into the environment, bind iron, and then re-enter the cell. Note: Lactoferrin and transferrin – proteins which can bind iron and limit bacterial growth (humoral barriers of innate immunity) 3/28/2021 Maha Fathy 22 D. Invasion and intra cellular survival 1. Escape intracellular killing by phagocytic cells and ability to intracellular growth e.g., • M. tuberculosis survives by inhibiting phagosome-lysosome fusion. https://www.sciencedirect.com/scien ce/article/pii/S1931312808001546 • Listeria spp. quickly escapes the phagosome into the cytoplasm before phagosome-lysosome fusion. 9/22/2019 https://www.researchgate.net/publication/326528987_Anti 23 microbial_Resistance_in_Listeria_Species/figures?lo=1 Maha Fathy Invasion and intra cellular survival (cont.) 2. Invasins: They are specific bacterial surface proteins that allow an organism to bind to specific receptors and invade normally non-phagocytic human cells e.g., invasins of Shigella spp. and Yersinia spp. Int. J. Mol. Sci. 2020, 21, 4102; doi:10.3390/ijm s21114102 , https://www.mdpi.com/14220067/21/11/4102/htm 9/22/2019 Maha Fathy 24 Invasion and extra cellular spread (cont.) 3. Spreading enzymes (extracellular spread): • Collagenase and hyaluronidase (allow the bacteria to spread through the tissues) e.g., S. Pyogenes • Lecithinase (breaks down lecithin of cell membrane) e.g., C. perfringens (considered as one of bacterial exotoxins) 9/22/2019 Maha Fathy 25 E. Type III secretion system (T3SSs or injectosomes) • They are syringe like protein appendages • Delivers bacterial toxins directly to the host cell. • They are found in many species of gram-negative bacteria e.g., E. coli and Pseudomonas, Salmonella, Shigella, Yersinia, Bordetella spp. https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC2176049/ 3/28/2021 Maha Fthy 26 F. Damage to host cells • Immune Mediated Damage • Bacterial Toxin 3/28/2021 Maha Fathy 27 Immune Mediated Damage Microbial antigens elicit immune response (humoral and cellmediated) resulting in immunopathology which may persist even after the infection is eliminated. • Cross-reaction of bacteria-induced antibodies with tissue antigens e.g., rheumatic fever following S. pyogenes infection • Delayed hypersensitivity and the granulomatous response e.g., cavitation in pulmonary tuberculosis and neurological damage in leprosy • Immune complexes damage: e.g., poststreptococcal glomerulonephritis 9/22/2019 Maha Fathy 28 Bacterial Toxins Effect Two Main Categories • Structural toxins (part of cell wall) • Secreted toxins (exotoxins) • Invasiveness and cell damage • Interfere with the host cell function • Trigger IR 9/22/2019 Maha Fathy 29 Structural Toxins • Endotoxin: (Lipid A portion of the outer membrane Lipopolysaccharides of gram-negative cell wall) which is the is primary virulence factor in gram negative associated sepsis & septic shock • Peptidoglycan and teichoic Acids: released when cells of gram-positive bacteria die can serve as structural toxins leading to chemotaxis and immune mediated reaction. 9/22/2019 Maha Fathy 30 Bacterial Exotoxins 9/22/2019 Maha Fathy 31 Exotoxins • They are toxic bacterial proteins, which may be excreted directly into the medium. • They are produced by a variety of bacteria including Gram positive and Gram-negative bacteria 3/28/2021 Maha Fathy 32 Structure and Functions of Exotoxin Types • A-B toxin • Membrane disrupting toxins • Super Antigens 3/28/2021 Maha Fathy 33 A-B Toxins • Diphtheria toxin • Pseudomonas exotoxin A • Cholera toxin 3/28/2021 Maha Fathy 34 Membrane Disrupting Toxins (Cytolytic enzymes) • Lyses host cells by disrupting the plasma membrane. • They can affect any host cell • Their in vitro action is obvious through hemolysis of RBCs (hemolysins) Examples • Lecithinase (phospholipase): C. perfringens. 3/28/2021 Maha Fathy 35 Superantigens • Non-specific (polyclonal) activation of very large numbers of CD-4 T lymphocytes • Release of large amount of interleukin-2 and other cytokines Toxic Shock (e.g. TSST-1 of S. aureus) 3/28/2021 Maha Fathy 36 Superantigen • Superantigen doesn’t need to be processed by antigen presenting cells • It binds to the variable segment of ß chain only (Vß) outside the TCR • Many different T cells with different antigen specificities become activated • Release of large amount of interleukin-2 and IFN-γ from T-cells (and other cytokines) Leads to Shock (Toxic Shock) • Example: TSST-1 (S. aureus) 3/28/2021 Maha Fathy 37 3/28/2021 Maha Fathy 38 Compare 3/28/2021 Exotoxins Endotoxins Source Chemistry Mechanism of release Antigenicity Effect of formalin Action Specificity Genetic Origin Toxicity Effect of heat Maha Fathy 39 Summary • Body of healthy individuals contain normal flora. • Host-Microbe Relationship are affected by both host factors and microbial factors. • Steps of bacterial pathogenesis: exposure, colonization, infection, disease production • Pathogenicity of microorganisms depends on their virulence factors • Bacteria have many virulence factors • Endotoxin or LPS is exclusive to, and an integral component of cell wall of Gramnegative bacteria • Exotoxins are toxic bacterial proteins that are produced by a variety of bacteria including Gram positive and Gram-negative bacteria. 3/28/2021 Maha Fathy 40 Compare and Contrast???? • Toxin & Toxoid • Septic shock & Toxic shock • Endotoxin & Exotoxin • A-B toxin & membrane disrupting toxin with giving examples 3/28/2021 Maha Fathy 41 Define • Colonization (as a step of bacterial pathogenesis) • Bacterial virulence factors • What are the different factors that help bacteria in their pathogenesis? • Describe how bacteria can escape (evade) IR? 9/22/2019 Maha Fathy 42 Most welcome for any question Click to edit Master title style • Edit Master text styles • Second level • Third level MahaFathy@med.asu.edu.eg • Fourth level • Fifth level 9/22/2019 Maha Fathy 43 Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Intended Learning Outcomes (ILOs) 1. Identify different components of bacterial cell genetic material. 2. Identify bacterial plasmids and compare it with transposons regarding structure and properties. 3. Describe structure and importance of bacteriophage. 4. Compare between bacteriolysis and lysogeny. 5. Compare between phenotypic and genotypic bacterial variation. 6. Recognize the differences between mutation and gene transfer. 7. Demonstrate differences between transformation, conjugation, and transduction. 8. Describe steps and applications of recombinant DNA technology. 26/2/2019 Maha Fathy 2 Genetic information in a bacterial cell are carried on: •Bacterial chromosome. •Plasmids. •Transposons. •Bacteriophages 26/2/2019 Maha Fathy 3 Bacterial Chromosome • Single molecule of double stranded DNA (double helix) • Each strand is a long sequence of nucleotide units • The bacterial chromosome carries the essential genetic materials (determinants) that control the bacterial properties and behavior Sugar Phosphate backbone Hydrogen bonds Base Pairs • It is a circular molecule free inside cytoplasm 10/17/2018 Maha Fathy 4 The 2 strands are complementary Replicates by semiconservative method 10/17/2018 Maha Fathy 5 Test your knowledge • What are the differences between bacterial and Eukaryotic chromosomes? 26/2/2019 Maha Fathy 6 Bacterial Plasmids • Extra-chromosomal circular double stranded DNA • They are capable of self replication (independent of cell replication) (autonomous) • They are dispensable (not mandatory) for life of the cell) • Two types: Transmissible (Conjugative) 26/2/2019 Non Transmissible (Non conjugative) Maha Fathy 7 Transmissible (conjugative) plasmids • Plasmids of Gram –ve bacteria carry genes coding for sex pili and enzymes required for transfer (tra gene) = F (Fertility plasmid) • Can transfer to other bacteria of the same or different species in a process called conjugation • They are usually present in a few (1–3) copies per cell (larger sized). What are the characteristics of plasmids of Gram negative bacteria? 26/2/2019 Maha Fathy 8 Non-transmissible (non-conjugative) plasmids • They do not contain the transfer genes • They are frequently present in many (10–60) copies per cell (smaller sized). Plasmids of Gram-positive bacteria are non transmissible e.g. plasmids that carry gene responsible for Beta lactamase production in Staphylococcus aureus. 26/2/2019 Maha Fathy 9 Test your knowledge •Differentiate between transmissible and Non transmissible plasmids 26/2/2019 Maha Fathy 10 Test your knowledge • Plasmids of Gram +ve bacteria are non conjugative (NO tra genes) How are they mobilized for transfer? 26/2/2019 Maha Fathy 11 What are the cell properties determined by plasmids? 1. Antimicrobial drug resistance 2. Virulence: ▪ Exotoxins production. ▪ Pili formation for adhesion. 3. Sex pilus formation for gene transfer (F or Fertility plasmid) 26/2/2019 Maha Fathy 12 Drug Resistance Genes coding for drug resistance for many bacteria are carried on plasmid (R-plasmid). 26/2/2019 Maha Fathy 13 What is R-plasmid? • R-plasmid is a transmissible plasmid (extrachromosomal DNA) carrying genes mediating antimicrobial resistance to different antibiotics Examples: • Chloramphenicol resistance gene • Penicillin resistance gene • Tetracycline resistance gene 26/2/2019 Maha Fathy 14 Transposons (jumping genes) • Several Kbp of DNA capable of mediating their own movement (jump or transpose) from one location to another on the same chromosome or from plasmid to plasmid or from chromosome to plasmid • They are non self replicon i.e. replicate with the other replicating DNA molecules e.g. plasmid or chromosome 26/2/2019 Maha Fathy 15 Effects of transposition •They can cause mutations in the gene into which they insert •They can alter the expression of nearby genes. 26/2/2019 Maha Fathy 16 Types of Transposons • Simple transposons (insertion sequence): They carry genes code only for enzymes responsible for its transposition (transposase) • Complex transposons They carry genes code for enzymes responsible for transposition plus other genes: e.g. • • Antibiotic resistance genes Toxin production genes. 26/2/2019 Maha Fathy 17 Insertion Sequence Complex Transposon 26/2/2019 Maha Fathy 18 Test your knowledge • How can transposons be transmitted from one bacterial cell to another? 26/2/2019 Maha Fathy 19 Bacteriophage • They are viruses that infect bacterial cells • They show high degree of host specificity 26/2/2019 Maha Fathy 20 Interaction between Bacteriophage and Bacterial Host Two types: • Bacteriolysis (Lytic cycle): by virulent or lytic phage • Lysogeny (lysogenic cycle): by temperate phage 26/2/2019 Maha Fathy 21 Bacteriolysis (Lytic cycle) • Infection of a bacterial host with a virulent phage leads to lysis of the bacterial host cell and release of new formed phages 26/2/2019 Maha Fathy 22 1. Adsorption of the phage to the bacterial host cell 4. Progeny phage assembly & release 2. Injection of the phage nucleic acid to the cytoplasm 3. Nucleic acid replication and synthesis of phage proteins 26/2/2019 Maha Fathy 23 Lysogeny (lysogenic cycle) • Infection of a bacterial host with a temperate phage leads to integration of phage DNA into the bacterial chromosome. • The integrated non lytic form which persists in the cell is called prophage (free in cytoplasm or integrated within chromosome. • The integrated prophage replicates within the bacterial chromosome and transferred to the progeny of the cell (without cell lysis) 26/2/2019 Maha Fathy 24 prophage replicates within the chromosome and transferred to the progeny of the cell Lysogenised cells Lysogenised cell Integration of the phage DNA into the bacterial chromosome 26/2/2019 Maha Fathy 25 NB In Lysogeny temperate phage can present either • Non integrated extra chromosome Or • Integrated into bacterial chromosome 26/2/2019 Maha Fathy 26 What is the result for the presence of this prophage? • Lysogenic conversion: The acquisition of new phenotypic bacterial cell characteristics coded by prophage e.g. • Toxigenicity of Corynebacterium diphtheria. 26/2/2019 Maha Fathy 27 What is the importance of Bacteriophages? • Lysogenic conversion (prophage) • Gene transfer: transfer of genetic information from one bacterial strain to another by transduction. • Bacterial Typing (phage typing): study of relatedness among bacteria • Genetic engineering (used as a vector) 26/2/2019 Maha Fathy 28 Bacterial Variation Phenotypic Genotypic • A reversible change in the observable structural and physiological properties of the cell without change in bacterial genes. • A change in the expression of genes e.g. Spore formation and vegetation • An irreversible change in the genome of a bacterial cell. • A change in genetic constitution • It can occur either by: • Mutation or • Usually in response to environmental condition • Gene transfer Define phenotypic or genotypic variation Compare between phenotypic & genotypic variation 26/2/2019 Maha Fathy 29 Compare between genotypic & phenotypic bacterial variation Phenotypic Genotypic • Reversible • Irreversible • Change in gene expression only • Change in gene constitution (mutation or gene transfer) • Not Heritable • Heritable • In response to environmental conditions • Spontaneous or induced 26/2/2019 Maha Fathy 30 Mutation • Error usually during replication of DNA leading to change in nucleotide sequence • Single base substitution (point mutation) • Frame- shift mutation (insertion and deletion) 26/2/2019 Maha Fathy 31 Mutation • Spontaneous (natural): during cell multiplication Or • Induced by exposure to physical agents (UV or gamma rays) or mutagenic chemicals (alkylating agents or nitrous acid) Can lead to antibiotic resistance 26/2/2019 Maha Fathy 32 Test your knowledge •Mention some applications on induced mutation with medical importance 26/2/2019 Maha Fathy 33 Gene Transfer •Transformation •Conjugation •Transduction 26/2/2019 Maha Fathy 34 Transformation (Uptake of pure “naked “DNA ) Donor Cell Recipient Cell Lysis Released DNA DNA taken up by another cell 26/2/2019 Maha Fathy DNA incorporated into recipient cell genome 35 What are the necessary conditions for transformation to occur? • Competence: Competent recipient cell • DNA Homology: There must be DNA homology between donor &recipient cells 26/2/2019 Maha Fathy 36 Does competence occur naturally or artificially? • Most bacteria are not naturally competent to be transformed by DNA. Why? • Bacterial cells contain restriction endonuclease enzymes which digest any foreign DNA. • In nature: in some bacteria competence factors are produced at a specific point in the growth cycle of bacteria that allows free DNA to cross bacterial the cell membrane • Competence can be induced artificially in the laboratory by treating cells with calcium chloride or heat shock which alter cell membrane permeability 26/2/2019 Maha Fathy 37 Conjugation (plasmid mediated) =horizontal transfer + + F+ F+ F Donor Recipient Plasmid Chromosomal DNA F Donor F Recipient Donor Plasmid Pilus unwinds formed one strand remains in the F+ cell while the other passes through the sex pili to the recipient F- cell 26/2/2019 Maha Fathy F Recipient A complementary strand is formed by both cells and both cells will become (F+) 38 Transduction (Bacteriophage) • It is the transfer of bacterial DNA from bacterium to another by a bacteriophage (bacterial virus)= horizontal transfer • Generalized transduction: • Phage nucleic acid is not integrated into bacterial chromosome (lytic or non integrated prophage) • Specialized transduction: • Phage nucleic acid is integrated into bacterial chromosome (integrated prophage) 26/2/2019 Maha Fathy 39 Generalized transduction can occur by virulent phage or non integrated prophage (any part of bacterial DNA can be picked & transferred) Bacterial cell containing both A & B genes The bacterial cell now become A+ve Phage carrying gene A Phage with gene A infects another or B 26/2/2019 bacterial cell (A Maha Fathy 40 –ve) Specialized transduction occur by integrated prophage (A specific part of bacterial DNA is picked & transferred) 26/2/2019 Maha Fathy 41 Test your knowledge Compare between Generalized transduction & Specialized transduction 26/2/2019 Maha Fathy 42 Recombinant DNA technology & Genetic Engineering • Genetic Engineering: Lab induced modification of the genotypes of organisms by incorporation of new genes from entirely different species • Recombinant DNA The DNA molecule which contains a new gene 26/2/2019 Maha Fathy 43 Recombinant DNA Vector Extraction (Bacterial plasmid) DNA extraction DNA of interest (specific gene) Both are treated with the same restriction enzymes Joining of the sticky ends of both the vector molecule and the specific DNA (ligase enzyme) Recombinant DNA 26/2/2019 Sticky (cohesive) ends Maha Fathy 44 Propagation (Gene Cloning) The recombinant DNA molecule is transferred to a bacterial cell by transformation. Cell multiply, creating many genetically identical bacteria (clones); each is able to produce the gene product (usually a protein). 26/2/2019 Maha Fathy 45 To summarize: Genetic engineering & recombinant DNA technology • Extraction of the wanted DNA sequence (specific gene) • Integration of this specific gene into a vector (plasmid or phage) to produce a recombinant DNA molecule • Propagation of the recombinant DNA molecule in a host organism for gene cloning and production 26/2/2019 Maha Fathy 46 Applications on genetic engineering and DNA recombination 1. Production of a substance (like a hormone or vaccine) 2. Production of probes for diagnosis of infectious diseases (hybridization reactions) 3. Gene therapy 4. Chromosome mapping and sequensing 26/2/2019 Maha Fathy 47 1. Production of many substances Like: Hormones (insulin) & Vaccines (Recombinant Hepatitis B vaccine). 26/2/2019 Maha Fathy 48 2. Hybridization reactions used for diagnosis The use of nucleic acid probe to detect complementary sequence of microbial genes in clinical samples. Molecular diagnosis for DNA detection without amplification 26/2/2019 Maha Fathy 49 3. Gene Therapy • It involves insertion of a normal gene into human cells to replace a defective gene to correct a specific genetic disorder (stem cell technology). 26/2/2019 Maha Fathy 50 To summarize Bacterial variation may be phenotypic or genotypic Genetic variation can occur in bacteria by mutation and gene transfer Mutation may be natural or induced Gene transfer can occur among bacteria by transformation, conjugation and transduction • Transformation: is the process by which DNA itself enters a recipient bacterium. • Conjugation: DNA (plasmid) is transferred directly from one bacterium (donor, F+) to another (recipient, F-) through sex pilus • Transduction: DNA, either plasmid or chromosomal, is transferred from one bacterium to another by a bacteriophage • • • • 26/2/2019 Maha Fathy 51 Self learning What do you know about CRISPR & CRISPR-Cas system in bacteria ? 26/2/2019 Maha Fathy 52 Most welcome for questions 4/3/2021 Maha Fathy 53 Click to edit Master title style • Edit Master text styles CLASSIFICATION AND CHARACTERIZATION • Second level OF MEDICALLY RELEVANT BACTERIA (Lecture 1) • Third level • Fourth level • Fifth level Dr. Makram F. Attalah Prof. of Medical Microbiology & Immunology Faculty of Medicine Ain shams University 1 Intended Learning Outcomes By the end of this lecture students should be able to: •Identify the approaches for classification of bacteria. •Identify the basis of phenotypic characterization of medically important bacteria. •Identify the basis of molecular biology and genetic composition of medically important bacteria. •Mention the important biological properties of medically relevant bacteria. 2 Classification of Distinguishing characteristics Physical properties of the cell wall Morphology Growth inside or outside host cell Reaction to Gram stain Shape Oxygen requirements 3 Methods of Classification Identification of Bacteria A. The old system of classification. B. The new system of classification. and 4 The old system of classification • • ✓ ✓ ✓ ✓ ✓ ✓ ✓ It was based on phenotypic characteristics. This system involves some important biological characteristic as: Morphology, Physical properties of cell wall, Shape, Reaction to Gram stain, Oxygen tolerance, Ability to form spores & Different enzymatic activities through different biochemical tests. 5 The new system of classification • It is based on molecular biology and genetic composition such as: 1. Nucleotide base composition [Genomic guanine (G) + cytosine (C) content]: The genomic G+C content in the total DNA is relatively fixed for any one species. 6 2. DNA base homology: The organisms are arranged into groups on the basis of the homology of their DNA base sequences (DNA Fingerprinting). • Living organisms that look different or have different characteristics also have different DNA sequences. • The more varied the organisms, the more varied the DNA sequences. • DNA fingerprinting is a very quick way to compare the DNA sequences of any two living organisms. 7 Medically Important Bacteria 8 9 10 Cocci ClickGram-Positive to edit Master title style Genus Staphylococci • Edit Master text styles Properties: • Second level • Staphylococci are Gram-positive cocci arranged in • Fourth level • Fifth levelirregular clusters (bunches). grape-like • All are catalase positive. • Third level 11 Click to edit Master title style Staphylococci classification and important species: Staphylococci are classified according to coagulase • Editproduction Master text styles into: • Second level 1.• Third Coagulase positive Stapylococci : Staphylococcus aureus level • Fourth level (S. aureus) • Fifth level 2. Coagulase negative Stapylococci (CoNS): Staphylococcus epidermidis & Staphylococcus saprophyticus. positive result positive result control drop 12 Staph aureus Click to edit Master title style Major characteristic • Edit Master text styles Beta-hemolytic. • •Second level • Third level • Coagulase-positive. • Fourth level • Fifthmannitol level • Ferments on mannitol salt agar. • Produce golden yellow endopigment. • Cell wall contains Protein A that binds Fc component of IgG and inhibits phagocytosis. 13 Epidemiology Click to edit Master title style colonizes: • EditItMaster text styles • Second level • Nasal mucosa (20-50 % of population). • Third level • Fourth level • Skin specially heath care workers. • Fifth level • Vagina (5%). • Can be found on contaminated environment e.g. clothing, bed linens, and fomites. • Acquisition of infection may be either exogenous or endogenous. 14 Diseases Click to edit Master title style • Edit Master text styles •• ThirdLocalized pyogenic infections. level Fourth level seated infections. • • Deep • Fifth level • Toxin-mediated diseases. • Second level 15 Localized pyogenictitle skinstyle infections Click to edit Master a furuncle, is a deep • Edit Master text styles folliculitis, • Second infection of level • Third level the hair follicle • Fourth level •furuncles Fifth level carbuncles A carbuncle is a cluster of boils, draining pus onto the skin impetigo, a common and highly contagious skin infection that causes sores and blisters. abscesses is a collection of pus that has built up within the tissue of the body surgical site infections impetigo 16 Special Program Students Wednesday 24/10/2018 Deep infections: Click to seated edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level osteomyelitis septic arthritis pyelonephritis pneumonia cellulitis mastitis empyema Special Program Students Wednesday 24/10/2018 renal abscess acute endocarditis 17 ClickToxin-mediated to edit Master title style diseases • EditTSST Master -1 text styles Epidermolytic toxin Enterotoxin • Second level • Third level Toxic • Shock Fourth level • Fifth level syndrome Scalded skin syndrome Special Program Students Wednesday 24/10/2018 Food poisoning 18 Staph. epidermidis Click to edit Master title style • EditMajor Master characteristics text styles • Second level • Coagulase-negative. • Third level • Fourth level • Non hemolytic. • Fifth level • Novobiocin sensitive. Special Program Students Wednesday 24/10/2018 19 Epidemiology Click to edit Master title style • Normal cutaneous flora. • Edit Master text styles • Opportunist, often by colonizing biomedical devices e.g. • Second level •intravascular Third level catheter, shunt, prosthetic devices. • Fourth level • Fifth level Diseases • Septicemias. • Endocarditis. • Wound infections. Special Program Students Wednesday 24/10/2018 20 saprophyticus ClickStaph. to edit Master title style • EditMajor Master characteristics text styles • Second level • Coagulase-negative. • Third level • Fourth level • Non hemolytic. • Fifth level • Novobiocin resistant. Special Program Students Wednesday 24/10/2018 21 Epidemiology Click to edit Master title style • Edit Master text styles • Urinary mucosa colonization. • Second level • Third level Diseases • Fourth level • Fifth level • Urinary tract infection, most commonly in sexually active young women (Honey moon cystitis). Special Program Students Wednesday 24/10/2018 22 Genus: Streptococcus (streptococci) Click to edit Master title style • EditImportant Master textproperties: styles • Second level • Streptococci are Gram-positive cocci arranged in chains or •inThird level pairs. • Fourth level • Fifth level • All Streptococci are catalase negative. Special Program Students Wednesday 24/10/2018 23 Streptococci classification Click to edit Master title style According hemolytic activity on sheep blood • Edit1.Master textto styles • agar: Second level Third (ᵝ) levelhemolytic: cause complete lysis of RBCs a. •Beta • Fourth level e.g. Streptococcus(S) pyogenes & S. agalactiae. • Fifth level b. Alpha (ᵅ) hemolytic: cause partial hemolysis of RBCs; producing greenish discoloration of blood agar e.g. viridans streptococci & S.pneumoniae. c. Non-hemolytic : have no effect on blood agar. 24 2. to Serological Classification: Click edit Master title style • Beta hemolytic Streptococci are further classified • Edit Master text styles into serogroups (A-O) according to carbohydrate (C) • Second level antigen • Third level (Lancefield classification) in their cell wall. • Fourth level • Fifth level Special Program Students Wednesday 24/10/2018 25 Click to edit Master title style • S. pyogenes • Edit Master text styles (Group A, beta • Second level hemolytic) are further • Third level serotyped through M • Fourth level • Fifth level protein (Griffith classification). • S. pneumoniae are serotyped according to capsular polysaccharides. 26 Streptococcus pyogenes Click to edit Master title style • EditMajor MasterCharacteristics text styles • •Second level Group A Streptococcal cell wall. • Third level • Beta-hemolysis. • Fourth level • Fifth level • Bacitracin-sensitive. • M protein in cell wall allows serological typing and it is an important virulence factor. 27 Epidemiology Click to edit Master title style • Found on the skin and in the oropharynx in small numbers. • Edit Master text styles Diseases • Second level Third level • •Pharyngitis/scarlet fever. • Fourth level • Fifth level • Impetigo/cellulitis/fasciitis/ erysipelas. • Post-infectious sequelae: acute glomerulonephritis and rheumatic heart disease. 28 Streptococcus agalactiae Click to edit Master title style • EditMajor Master Characteristics text styles • Second level • Group B cell wall carbohydrates. • Third level • Fourth level • Beta-hemolytic. • Fifth level • Bacitracin resistant. 29 Epidemiology Click to edit Master title style • Edit• Master text styles Commensal in the vagina of some females, it colonizes • Second 15% level to 20% of pregnant women. • Third level • Newborn • Fourth level infected during birth (increased risk with • Fifth level prolonged labor after rupture of membranes). Diseases • Neonatal septicemia, pneumonia, and meningitis. 30 Viridans streptococci e.g. Click to edit Master title style S. mutans • EditCharacteristics Master text styles • Second level oral • Normal flora. • Third level • Alpha-hemolytic. • Fourth level • Fifth level • Not inhibited by optochin. • Not lysed by bile. Turbid bacterial suspension Turbid bacterial Suspension after addition of sodium deoxycholate Reagent (bile salt) Special Program Students Wednesday 24/10/2018 31 Click to edit Master title style • EditEpidemiology Master text styles • Second level • Members of the resident flora of the oral cavity. • Third level Diseases • Fourth level • Fifth level • Major role in dental caries. • Subacute bacterial Endocarditis. Special Program Students Wednesday 24/10/2018 32 Streptococcus pneumoniae Click to edit Master title style Characteristics • Edit Master text styles • Second level • Alpha-hemolytic. • Third level • Sensitive • Fourth levelto optochin. • Fifth level • Lysed by bile. Turbid bacterial suspension viridan s Clear bacterial Suspension after addition of sodium deoxycholate Reagent (bile salt) Special Program Students Wednesday 24/10/2018 33 Epidemiology Click to edit Master title style • 5%–50% of the healthy population harbors virulent organisms in the oropharynx. • Edit Master text styles • Second levelresults most often when predisposing factors are present • Disease •e.g. Thirdheavy level smoking, alcohol intoxication, previous viral respiratory • Fourth level tract infections and splenectomy. • Fifth level Diseases • Lobar pneumonia. • Otitis media. • Sinusitis. • Meningitis in adults. Special Program Students Wednesday 24/10/2018 34 faecalis ClickEnterococcus to edit Master title style Enterococcus faecium • Edit Master text styles • Second level • Third level Characteristics • Fourth level • Fifth • Group D level streptococcal cell wall carbohydrates. • Salt and bile tolerance. • High pattern of antibiotic resistance. Special Program Students Wednesday 24/10/2018 35 Click to edit Master title style Epidemiology • Their natural habitat is in the human intestines. • Edit Master text styles • Theylevel may cause diseases outside the • Second •gastrointestinal Third level tract particularly in hospitalized or • Fourth level immunocompromised patients. • Fifth level Diseases • Endocarditis. • Septicemia. • Urinary tract infections. Special Program Students Wednesday 24/10/2018 36 Peptostreptococcus Click to edit Master title style • Edit•Anaerobic Master text stylesstreptococci. • Second level •Opportunists. • Third level • Fourth level • Fifth level Special Program Students Wednesday 24/10/2018 37 Gram-Positive Cocci Click to edit Master title style Differentiation of Medically Important Gram Positive Cocci • Edit Master text styles • Second level • Third level • Fourth level • Fifth level R S Special Program Students Wednesday 24/10/2018 38 Gram Positive Click to edit Master title styleBacilli • EditNon Masterspore text styles • Second level forming • Third level • Fourth level • Fifth level Corynebacteria Listeria Special Program Students Wednesday Spore forming Aerobic Anaerobic Bacillus group Clostridia group 39 Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Special Program Students Wednesday 24/10/2018 40 Genus : Bacillus Click to edit Master title style Important members • Edit Master text styles • Second level • Third level • B anthracis • Fourth level • Major Characteristics • Fifth level • Aerobic spore forming large Gram positive bacilli occurring in chains • Have polypeptide capsule (poly-d- glutamate) Special Program Students Wednesday 24/10/2018 41 41 ClickEpidemiology to edit Master title style •Most members of the genus bacillus are saprophytic • Editorganisms Master textinstyles soil, water and air. • Second •Few level are pathogenic • Third level •contact • Fourthwith level infected animals or inhalation of spores (zoonotic) • Fifthbioterrorism level •Potential agent •Diseases •Anthrax disease •-Cutaneous (malignant pustule) •-Inhalation (wool sorters’ disease) •-Intestinal (rare). Special Program Students Wednesday 24/10/2018 Special Program Students Wednesday 24/10/2018 42 42 Click edit Master title style B. to Cereus • EditEpidemiology Master text styles • Second level Saprophytic found in nature • Third level • Fourth level • Fifth level •Diseases • Food poisoning • Associated with food kept warm, e.g. Chinese fried rice • Eye infections (rare) Special Program Students Wednesday 24/10/2018 Special Program Students Wednesday 24/10/2018 43 43 Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Special Program Students Wednesday 24/10/2018 44 Click to edit Master title style • Edit Master text styles CLASSIFICATION AND CHARACTERIZATION OF MEDICALLY RELEVANT BACTERIA (2) • Second level • Third level • Fourth level • Fifth level Dr. Wafaa khalil zaki Prof. of Medical Microbiology & Immunology Intended Learning Outcomes By the end of this lecture students should be able to: Mention the important biological properties of : • Gram-positive bacilli : clostridium ,Corynebacterium ,Actinomyces, Nocardia . • Gram negative cocci N. meningitids & N. gonorrheae • Gram negative rods (Enterobacteriaceae) • Gram negative rods (Non fermenting aerobic )pseudomonas . • Curved Gram negative Bacilli : Vibrio ,Campylobacter, Helicobacter. Gram Positive bacilli Aerobic non spore forming Gram-positive bacilli Click to edit Master title style Listeria Corynebacterium diphtheriae ➢ Aerobic, • EditCharacteristic Master text stylesGram positive bacilli, non- • ➢ Short, Gram positive, , non spore forming bacilli. Motile , distinguish from diphtheria by motility . spore forming, club shaped (with Second level one swollen end and another • Third level tapering end) , Chinese letters like arrangement, non motile • Fourth level ➢ It is found in G.IT of animals, transmitted by • Fifth level Man is the only reservoir (case or unpasteurized milk, Dairy products (zoonosis) carrier) transmission: droplet (main) and contact Diseases Diphtheria Listeriosis Click to edit Master title style CLOSTRIDIUM Gram-positive , spore forming large • Edit Master styles . All members are bacilli,text Anaerobic motile and non capsulated • Second level • Third level EXCEPT C. perfringens. • Fourth level • Fifth level The organisms are found in the intestine of man, animal, and soil. Pathogenicity of the organisms due to exotoxins production tetani ClickClostridium to edit Master title style (Cl. tetani ) • Edit Master stylesanaerobic, • They aretext large, • Gram-positive, Second level • Third level spore- forming bacilli. • Fourth level • Fifth level • Produces tetanus toxin • Their natural habitat is the soil and the lower intestinal tract of man and animals • Transmitted through puncture wounds/trauma • (requires low tissue Oxygenation) Cl.toperfringens Click edit Master title style • Nonmotile “stormy fermentation” in milk • Edit Master text styles media • Second level •• Double Third level zone of • Fourth level hemolysis • Fifth level ➢food borne and traumatic implantation . ➢The main cause of gas gangrene and can cause food poisoning Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Cl. botulinum Click to edit Master title style ➢Produces botulinum toxin • Edit Master text styles food borne •➢Transmission: Second level • Third level ➢Food poisoning (botulism) • Fourth level • Fifth level Clostridium difficile Click to edit Master title style • Edit Master text styles • Second level • ThirdAntibiotic level • associated Fourth level • Fifth level pseudomem branous colitis Click to edit Master titleforming style Gram positive Filamentous non spore • Edit Master text styles bacilli • Second level • Third level • Fourth level • Fifth level Aerobic: Nocardia Anaerobic: Actinomyces ACTINOMYCETES Click to edit Master title style Actinomyces israelii • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Anaerobic Gram-positive bacilli that tend to form long branching filaments, Non−acid fast Nocardia asteroides Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level • It is strict aerobic Gram-positive bacilli that form branched filaments. • It is weak acid fast (5% sulphuric acid). weak acid fastness, aerobic growth differentiate Nocardia from Actinomyces. ACTINOMYCES NOCARDIA Click to edit Master styleStrict Aerobic; Gram-positive branching bacilli, - Anaerobic , title Gram-positive Characteristic Branching bacilli, Non−acid fast - Most members are normal • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Diseases flora colonize the mouth, upper respiratory tract, gastrointestinal tract and female genital tract that can cause disease when the normal mucosal barrier is disrupted actinomycosis; draining abscesses Endogenous infection weakly acid-fast. - They are environmental saprophytes that cause disease in immunocompromised individuals (opportunistic pathogens). Nocardiosis ❑ N. asteroides cause bronchopulmonary nocardiosis. ❑ N. brasiliensis cause Actinomycetoma Gram Negative Cocci ClickGenus: to edit Master title style Neisseria • Edit Master text styles and species: Important properties • •Second level are oxidase positive aerobic Gram Neisseria • Third level diplococci; occurring as pairs with negative • Fourthadjoining level flattened sides. • Fifth level • All ferment glucose. • They colonize mucosal surfaces (both pathogenic and non pathogenic). • The pathogenic members of the group includes N. meningitidis) is capsulated and maltose fermenter however, N. gonorrheae is not capsulated and non –maltose fomenter causes gonorrhea. 15 Negative Rodsstyle (bacilli) ClickGram to edit Master title Enterobacteriaceae • Edit Master text styles (Fermenting Gram Negative •Bacilli) Second level • Third level • This• is a large family of fermenting Gram-negative rods that Fourth level • Fifth level includes many genera as: Escherichia, Klebsiella, Enterobacter, Proteus, Salmonella, Shigella & Yersinia. 16 Natural Habitat: Click to edit Master title style • The majority are present as the normal flora in the colon text ofstyles both man and animals (e.g. E. coli • Edit Master • Second level ,Klebsiella ). • Third level • Some are • Fourth level saprophytes in water, soil and plants (e.g. • Fifth level Proteus a cause of UTI . • Few of them are primarily intestinal pathogens (e.g. Salmonella & Shigella). 17 Click to edit Characteristics Master title style General of Enterobacteriaceae Family: • Edit Master text styles • Theylevelare • Second Gram-negative bacilli. non spore forming, •non-capsulated Third level (with few exceptions). • Fourth level Fifth level • They• are motile with peritrichate flagella (with few exceptions). • They can grow on ordinary media as well as on selective and differential media e.g. MacConkey’s agar . 18 Click to edit Master title style All members share 4 common characteristics: 1. Facultative • Edit Master text styles anaerobe. • Second level 2. Oxidase negative. • Third level • Fourth level 3. Reduce nitrate to nitrite. • Fifth level 4. Ferment glucose (either ┴ or ┼). 19 Antigenic structure O,H &K Click to edit Master title. style ➢ O antigen (somatic antigen ) it is the outer polysaccharide • Edit Master text styles portion of the lipopolysaccharide. • Second level • Third level Fourth level ➢ H• antigen is the flagellar antigen . • Fifth level ➢ K antigen is the capsular antigen . Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Click to edit Master Escherichia coli title style • Edit• Master Motiletext andstyles Lactose • Second level fermenter • Third level • Indole positive • Fourth level • Fifth level • Some strains are capsulated • Major component of normal colon flora, may colonize vagina or urethra • Can be acquired endogenous or exogenous through contaminated hands of HCWs • Diarrheagenic strains may be transmitted through contaminated water or food Special Program Students Sunday 28/10/2018 Click to edit Master title style Causetext diseases • Edit• Master styles inside & outside GIT • Mostlevel common cause of urinary tract infection • Second Third level meningitis • •Neonatal • Fourth level • Common of different health care associated infections • Fifthcause level (UTI, SSI, Septicemia, VAP*) • Some strains can cause diarrhea (Diarrheagenic E. coli) Klebsiella spp. title style Click to edit Master • Edit•Master text styles Capsulated, non-motile • Second level • •Lactose Third level fermenting with mucoid • Fourthcolonies level • Fifth level • Important species include: K. pneumonaie (indole negative) & K. oxytoca (indole positive) • Colonizes human colon and upper respiratory tract • Can be acquired endogenous or exogenous Special Program Students Sunday 28/10/2018 Click to edit Master title style DISEASES • Edit• Master styles outside GIT Cause text disease • Second only level • Third level • Community acquired • Fourth level pneumonia Lander • Fifth level (Fried Pneumonia) • Neonatal sepsis • Common cause of different health care associated infections (UTI, SSI, IVDRIs, VAP*) spp. title style ClickProteus to edit Master • Highly motile (swarming growth)text styles • Edit Master • •Second level Non lactose fermenter • Third level • Have rapid • Fourth levelurease activity • Fifthmembers level • Important include: P. mirabilis (indole negative) and P. vulgaris (indole positive) Click to edit Master title style instyles human colon and environment (water and • Edit✓Found Master text soil)level • Second • Third level ✓Can be acquired endogenous or exogenous • Fourth level • Fifth level ✓Cause disease outside GIT only ✓UTI (associated with urinary stone formation) ✓cause of different health care associated infections (UTI, SSI, IVDRIs, VAP*) Salmonella spp. Click to edit Master title style Non lactose, Non sucrose fermenter • Edit• Master text styles • Motile • Second level Third level • •Important members: • Fourth level − S. enterica (typhi & paratyphi serovars) • Fifth level − S. cholerasuis − S. enteritidis & typhimurium • Human is the only reservoir for S. typhi (person to person spread) Special Program Students Sunday 28/10/2018 DISEASES Click to edit Master title style • Cause diseases inside & outside GIT • Edit Master text styles • Enteric • Second level(Typhoid fever) caused by S. enterica level suis cause bacteremia with focal lesions (including • •S.Third cholera • Fourth level osteomyelitis and arthritis) • Fifth level • Enterocolitis (S. enteritidis & typhimurium) Shigella spp. Click to edit Master title style • Non motile , Non lactose fermenter • Edit Master text styles • Reservoir: human colon only (no • Second level carriers) •animal Third level • Fourth level • Transmission: fecal-oral spread, • Fifth level person to person Cause disease inside GIT. • Bacillary dysentery (inflammatory bloody diarrhea) Yersinia spp. Click to edit Master title style • Medically important members: • Edit Master text styles • Second level −• Y.Third pestis level Fourth level − Y. •enterocolitica • Fifth level Transmitted from animals (zoonotic) Y. pestis causes plague Y. enterocoliica causes diarrhea or pseudoappendicitis (right lower abdominal pain due to mesenteric adenitis and/ or terminal ileitis). Special Program Students Sunday 28/10/2018 Non- Fermenting Aerobic Gram Negative Bacilli Click to edit Master title style Genus: Pseudomonas Important properties: • Edit Master text styles • Theylevel are aerobic Gram-negative motile • Second polar flagella . Strict aerobes. •with Third level • Fourth level • They •do Fifthnot level ferment sugars, they utelize glucose by oxidation . • They are oxidase-positive • They produce water soluble bluish-green exopigments (pyocyanin & pyoverdine). 32 Habitat and Transmission Click to edit Master title style • P. aeruginosa is widely distributed in nature in soil, water, plants and animals. • Edit• Master text styles Also inhabits the skin, upper respiratory • Second tract,level and colon of about 10% of people. • Third level • Its •ability to grow in solutions has resulted Fourth level in contamination of hospital respirators, • Fifth level humidifiers, anesthesia equipment, intravenous fluids, and even distilled water. • Pseudomonas has a remarkable ability to withstand disinfectants and can grow in antiseptics, and in detergents. 33 Diseases Click to edit: Master title style It causes diseases in man with abnormal host defenses • Edit(opportunistic Master text styles pathogen) and it is a major cause of hospital • Second level infections: acquired • Third level • Fourth level • Nosocomial pneumonia (most common cause of • Fifth level ventilator associated pneumonia), Sepsis, and urinary tract infections primarily in patients with lowered host defenses. • Chronic lower respiratory tract infections in patients with underlying disease e.g. cystic fibrosis. • Wound infections in sever burn patients. 34 Curved Gram Negative Bacilli Click to edit MasterGenus: title Vibrio style • This genus includes more than 30 species that are commonly found in aquatic environments. • Edit Master text styles The level most important Vibrio species is Vibrio cholerae; the • •Second agent of cholera disease. •causative Third level • Another important member is V. • Fourth level medically • Fifth level parahemolyticus which causes gastroenteritis due to consumption of fresh or under cooked see food. Click to edit Master title style CAMPYLOBACTER • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Important properties and species: Click to edit Master title style − Campylobacters • Edit•Master text styles are curved, Gram-negative rods that appear either comma or S shaped. • Second level • They Third level are microaerophilic, requires 10% CO2 • Fourth level for growth. Most important species are • Fifth level Campylobacter jejuni & Campylobacter coli. C. jejuni grows well at 42°C. • − Domestic animals such as cattle, chickens and dogs carry the organism in their intestinal tract and serve as a source of infection for humans (zoonotic disease). Click to edithabitat Master title style Natural • Edit Master text styles • Second level Third level It• exist as normal gastrointestinal tract flora of many • Fourth level animals e.g. • Fifth level cattle, chickens, and dogs (zoonotic disease). Transmitted by: Click to edit Master title style • 1. Ingestion of contaminated food (such as undercooked • Edit Master textor styles poultry meat) and drinks (as unpasteurized milk, • Second level contaminated water). • Third level • 2. •Contact Fourth level with infected animals or animal products. Fifth level • They• are common human pathogens, causing mainly: • 1. Enteritis • 2. Occasionally systemic infection. • Infections of campylobacter can be complicated by Guillain-Barré syndrome Genus: Helicobacter Click to edit Master title style -Helicobacters are curved Gram-negative rods and appear spiral in tissue biopsies. • Edit Master text styles − They are microaerophilic and have rapid • Second level urease activity • Third level • Fourth level − H. pylori is the causative agent of • Fifth level gastritis and peptic ulcer disease -Infection with H. pylori is a risk factor for gastric carcinoma and is linked to mucosalassociated lymphoid tissue (MALT) lymphomas. Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Click to edit Master title style • Edit Master text styles CLASSIFICATION AND CHARACTERIZATION OF MEDICALLY RELEVANT BACTERIA (3) • Second level • Third level • Fourth level • Fifth level Dr. Wafaa khalil zaki Prof. of Medical Microbiology & Immunology Intended Learning Outcomes By the end of this lecture students should be able to: Mention the important biological properties of : • Gram-Negative Rods Related to the Respiratory Tract: Hemophilus, Bordetella and Legionella. • Bacteria causing zoonotic diseases: Brucella, Pasteurella and Francisella. • Anaerobic Gram Negative Bacilli: Bacteroides. • Bacteria Not stained By Gram Stain: Mycobacterium and Spirochetes. • Cell Wall Free Bacteria: Mycoplasma. • Obligate Intracellular Bacteria: Chlamydia and Rickettsia. Gram-Negative Rods Related to the Respiratory Click to edit Master title style Tract There text are styles three medically important Gram-negative rods • Edit• Master typically • Second level associated with the respiratory tract: level 1.• Third Hemophilus influenzae (H. influenza), • Fourth level • Fifth level pertussis (B. pertussis), 2. Bordetella 3. Legionella pneumophila (L. pneumophila). • H. influenzae and B. pertussis are found only in humans, whereas L. pneumophila is found primarily in environmental water sources. ClickGenus: to editHemophilus Master title style • Edit Master textproperties: styles Important • Second level • It• Third is level a group of short Gram • Fourth level negative bacilli. • Fifth level • Requires enriched media, usually containing blood or its derivatives, for its isolation. (It needs growth factors called X (Hemin) and V (NAD) factors). Click to edit Master title style • Edit •Master stylesvirulent The text most strains of H. influenzae • Second level polysaccharide capsule with six • have Third level a • Fourth level serotypes, (a-f). • Fifth level • Type (b) is the most virulent. • Other strains are not encapsulated. ClickDiseases to edit Master title style H. influenzae • Edit•Master text styles type b causes meningitis and • Second level • Third level epiglottitis in infants and • Fourth level • Fifth level children. • Non-capsulated strains usually cause non invasive infections e.g. otitis media and sinusitis. First year .infection module ClickGenus: to editBordetella Master title style properties: • EditImportant Master text styles • Second level •• Gram Third level negative coccobacilli. • Fourth level • Fifthimportant level •Most species is: Bordetella pertussis, the causative agent of pertussis (whooping cough). Firs year. infection module Genus: Legionella Click to edit Master title style Important properties: • Edit Master text styles • It is a large family comprising many different • Second level •species. Third level • Fourth level • Fifth level • L. pneumophila is the pathogen of greatest medical importance. • Legionella is a facultative intracellular pathogen. Diseases: Legionellosis (Legionnaire disease). Special Program Students Sunday 28/10/2018 Click• Habitat to editisMaster title stylewater in environmental sources. • They are by and survive within • Edit Master text engulfed styles free-living amoebas which act as a • Second level • reservoir. Third level • Fourth level Transmission: • Fifth level • Inhalation or aspiration of aerosols containing the bacteria generated from contaminated air conditioning systems, water-cooling towers, water taps, and showerheads. • No person to person transmission. Click to edit Master title style Genus: Brucella Important properties: • Edit Master text styles • Brucella • Second levelspecies are small, coccobacillary, Gram-negative rods. Third level • •Morphologically they resemble Hemophilus and Bordetella. • Fourth level Disease:• Fifth level The causative agent of Brucellosis (undulant fever- Malta fever). Transmission: • Brucellosis is a true zoonotic disease i.e. all infections are transmitted to humans from animals. • No human to human infection. Pasteurella multocida Click to edit Master title (P.multocida) style • Edit Master text styles Important properties: • Second level level It• isThird a short, encapsulated Gram negative • Fourth level • Fifth level rod that exhibits bipolar staining. Transmission: The organism is part of the normal flora in the mouths of many animals, particularly domestic cats and dogs, and is transmitted by biting. Click to edit Master title style • EditDisease: Master text styles • Second level • •ItThird level causes wound infections • Fourth level with cat and dog bites. associated • Fifth level • A rapidly spreading cellulitis at the site of an animal bite. • Osteomyelitis can complicate cat bites. First year .infection module Anaerobic Gram Negative Bacilli Click to edit Master title style Genus: Bacteroides Important properties: • Edit Master text styles They are anaerobic Gram negative bacilli. •• Second level • They arelevel part of the normal flora. • Third • Fourthare level endogenous, usually arising from a • Infections • Fifth break in a level mucosal surface, and are not communicable. • Bacteroides fragilis is the most frequent pathogen. Disease: • Members of the genus Bacteroides are the most common cause of serious anaerobic infections (e.g., sepsis, peritonitis, and abscesses). Bacteria Not stained By Gram Stain Click to edit Master title style Mycobacteria • Edit Master text styles Important properties: • Second level • The most • Third level important characteristic of Fourth level is the property of acidthis • genus • Fifth level fastness, i.e. they are difficult to stain because of their thick cell wall and its high lipid content, but once stained, they resist decolonization by acid or alcohol and are therefore called acidfast bacilli (AFB). Click to edit Master title style Medically important members of the genus Mycobacteria: • Edit Master text styles • Second level Mycobacterium • Third level tuberculosis complex • Fourth level (MTB), Mycobacterium leprae • Fifth level Mycobacterium tuberculosis Tuberculosis in man. Human is the only reservoir. Atypical mycobacteria Or Mycobacteria other than tuberculosis (MOTT) or Non tuberculous mycobacteria (NTM) Leprosy Mycobacterium bovis may be associated with human disease. Tuberculosis in cattle, may be transmitted to man causing tuberculosis Mycobacterium tuberculosis Click to edit Master title style (M. tuberculosis) Important properties: • Edit Master text styles Theylevel are slender, straight or curved • •Second • Third level rods arranged singly or in pairs, • Fourth level non-motile, non-capsulated and • Fifth level non-spore forming. • They grow in parallel groups called cords due to presence of a cord factor (trehalose dimycolate) which is an important virulence factor. Special Program Students Sunday 28/10/2018 Cord Factor • M. tuberculosis grows Click to edit Master title style slowly (i.e., it has a doubling time of 18 hours) • Edit Master text styles • Second level • •Mycobacterial cell wall is Third level characterized by presence • Fourth level Fifth level of •high lipid content (mycolic acids) which permits intracellular survival and doesn’t allow staining by Gram’s stain. Mycolic Acid Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level ZN STAIN Click to edit Master title style are • Edit•They Master text stylesstained by Ziehl Neelsen (ZN) • Second level stain. • Third level • Fourth level tuberculosis. Disease: • Fifth level ZN stained film Clinical forms of tuberculosis: Click to edit Master title style • Pulmonary TB: it is the most common clinical form. Extrapulmonary • Edit• Master text stylesTB: affecting other organs e.g. meninges, kidney or bone.level • Second Thirdtuberculosis. level • •Skin • Fourth level • Milary• TB: is a wide disseminated form. Fifth it level Click to edit Master title style of styles transmission: • EditMode Master text • Second level by air borne particles that arise from patients • Third level level with• Fourth open pulmonary tuberculosis or skin • Fifth level lesions. Click to edit Master title style Mycobacterium leprae • Edit Master text styles Important properties: • Second level Third level • •Acid fast bacillus non motile non • Fourth level capsulated • Fifth level and non-spore forming. • A primary human pathogen. • M. leprae has not been grown in the laboratory; either on artificial media or in cell culture. Special Program Students Sunday 28/10/2018 • The optimal temperature for growth (30°C) is Clicklower to edit Master title style than body temperature. Disease: Leprosy • Edit Master text styles in man. ••Second level granulomatous disease affecting the A chronic • Third mucous level skin, membrane, and peripheral nerves • Fourth level (the lesions • Fifth level involve the cooler tissues of the body). Mode of transmission of leprosy: • Infection is acquired by prolonged contact with patients with lepromatous leprosy, who discharge M. leprae in large numbers in nasal secretions and from skin lesions. Special Program Students Sunday 28/10/2018 The face of a man with active lepromatous leprosy Disease Click to edit Master title style cell • EditThe Master textmediated styles immunity of the patients defines the clinicopathological picture which are Tuberculoid • Second level • Thirdof levelleprosy and Lepromatous type of Leprosy: type • Fourth level • Fifth level • Tuberculoid type: This is the mild form of leprosy in which cell mediated immunity (CMI) is intact. • Lepromatous type: It is the severe form of the disease in which CMI is depressed. Therefore, M. leprae grows in the skin and superficial nerves Special Program Students Sunday 28/10/2018 Spirochetes Click to edit Master title style Heterogeneous group of spiral motile • Editbacteria. Master text styles • Second level • They include 3 genera: • Third level • Fourth level 1. Treponema pallidum (T. pallidum) causes • Fifth level syphilis (STD). 2. Leptospira: Cause leptospirosis, a zoonotic disease transmitted by animal urine in water. 3. Borrelia: Cause Lyme disease (Borrelia burgdorferi) and relapsing fever (other Borrelia species). Special Program Students Sunday 28/10/2018 Cell Wall Free Bacteria ClickMycoplasma to edit Master title style General characters : • Edit Master text styles • The smallest free-living organisms. • Second level • Prokaryotic cells resemble Gram • Third level negative • Fourthbacteria. level • Fifth level • Their outer surface is a flexible cell membrane containing cholesterol but lack a cell wall. • So, these organisms can assume a variety of shapes (pleomorphic). Click to edit Master title style • Stain poorly with Gram • Edit Master styles stain, text but stain well with • Second level Giemsa’s stain or Leishman • Third level stain. • Fourth level level • Are • Fifthnot sensitive to antibiotics that act on cell wall e.g. ᵝ-lactam antibiotics. • Are not destroyed by lysozymes. Giemsa’s stain Click to edit Master title style specie associated with human disease: • EditThree Mastermycoplasma text styles • Second level • Mycoplasma • Third level pneumoniae: which is the cause of atypical pneumonia. • Fourth level • Fifth level • Mycoplasma hominis: has been implicated as an infrequent cause of pelvic inflammatory disease. • Ureaplasma urealyticum: may cause approximately 20% of cases of nongonococcal urethritis. Obligate Click to edit MasterIntracellular title style Bacteria Chlamydia • EditGenus: Master text styles General • Second levelCharacters: Third level • •Non motile coccoid bacteria. • Fourth level • Fifthobligate level • They are intracellular pathogen because: They lack the ability to produce sufficient energy and cannot synthesize ATP to grow independently and therefore can grow only inside host cells. • Don’t grow on artificial bacteria culture media. • Grow on yolk sac of chick embryo or in tissue culture. Click to editcaused Master style Disease bytitle Chlamydia: • EditChlamydia Master text trachomatis: styles • Second level • Types A, B, Ba, and C: cause Trachoma. • Third level • Types D–K: • Fourth level cause inclusion conjunctivitis & nongonococcal • Fifth level urethritis. • Types L1-L3: cause Lymphogranuloma venereum. Chlamydia pneumoniae causes atypical pneumonia. Chlamydia psittaci causes psittacosis (atypical pneumonia). Click to edit Master title style Genus: Rickettsia General Characters: • Edit Master text styles • Small, aerobic, coccobacilli. • Second level Third level intracellular parasites of eukaryotic cells. • •Obligate • Fourth level • May reside in the cytoplasm or within the nucleus of the cell that • Fifth level they invade. • Have typical Gram-negative cell walls. • All are maintained in animal & arthropod reservoirs. • Transmitted by arthropod vectors (e.g., ticks, mites, lice or fleas). • Humans are accidentally infected. Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level General Mycology Intended Learning Outcomes (ILOs) 1. Explain the eukaryotic nature of the fungi. 2. Define: fungal spores, yeast, filamentous fungi, dimorphic fungi, and other scientific terminology peculiar to mycology 3. Explain how fungi cause diseases. 4. Correlate the peculiar structures in fugal cells and mode of action of antifungal agents 5. Discuss the basis of classification of medically-important fungi. 6. Outline the underlying mechanisms of antifungal resistance General Characters of Fungi •Eukaryotic cells that have a true nuclei •Devoid of chlorophyll •Can absorb all nutrients from the environment specially decaying matter •Grow better at acidic pH Eukaryotic Prokaryotic Example Fungi, human cell bacteria Nuclear material True nucleus Nuclear membrane Nucleoli Multiple chromosome Histones Single chromosome No histones Mitosis Simple binary fission Division Cytoplasmic structure Mitochondria Microtubular cytoskeleton RES Ribosome : 80S Absent Absent 70S Cytoplasmic membrane Contains sterol No mesosomes Contain lipoprotein No sterol Mesosome lipoprotein Cell Wall fungi contain chitin, mannan, glucan Present (except mycoplasma) © Fungi are present as • Saprophytes in nature: Most species are beneficial. They are essential in breaking down and recycling organic matter. Some fungi greatly enhance the quality of life by contributing in production of food as bread and cheese. Other fungi produce useful bioactive metabolites as antibiotics (e.g penicillin) and cytotoxic drugs as cyclosporine • Commensal: Live in harmony on humans, driving their nutrition from compounds on body surfaces. Some are opportunistic under certain conditions they may invade tissues or vasculature and cause disease • Pathogenic: Infect the healthy and cause severe disease in the immunocompromised •There are over 100,000 species recognized, with 100 infectious agents to humans. •Moulds are composed of numerous, microscopic, branching hyphae known collectively as a mycelium. •Yeasts are composed of fungal cells giving the appearance of smooth colonies •Dimorphic fungi © Fungal spores: • Fungal spores are microscopic biological particles forming a part of the life cycle of fungi, allowing fungi to be reproduced. Like bacterial spores, they have an enhanced survival value and structural features that promote spread. •Airborne fungal spores are one of the major causes of respiratory illness in humans. • The presence/absence of spores and their size, shape and location are major features used in the laboratory to identify the species of fungus in clinical specimens. Overview of fungal diseases Fungi may cause diseases by one of the following mechanisms: 1- Allergies: Many fungal spores contain allergens which can trigger a range of respiratory symptoms in those susceptible, including sneezing, runny nose, mucous production, cough, congestion, sinusitis, earache, headache, and wheezing. 2- Mycotoxicosis • Mycotoxins are secondary metabolites produced by fungi and are capable of producing diseases. • The mycotoxicosis is a food poisoning caused by ingestion of food containing a sufficient quantity of mycotoxins. Mycotoxicoses, like all toxicological syndromes, can be acute with rapid onset or chronic characterized by low-dose exposure over a long time period, resulting in cancers and other generally irreversible effects. • Chronic intoxication occurs worldwide, but especially in climates with high temperature and humidity and where grain is harvested with high water content. QUIZ Regarding the structure and reproduction of fungi, which one of the following is most accurate? (A) Peptidoglycan is an important component of the cell wall of fungi. (B) Molds are fungi that grow as single cells and reproduce by budding. (C) Some fungi are dimorphic (i.e., they are yeasts at room temperature and molds at body temperature). (D) The fungal cell membrane contains ergosterol, whereas the human cell membrane contains cholesterol. (E) As most fungi are anaerobic, they should be cultured under anaerobic conditions in the clinical laboratory. • Mycoses : disease caused by infection with a fungus that may cause superficial, cutaneous, subcutaneous, or systemic diseases : a. Superficial skin infections: Infections in stratum corneum b. Cutaneous mycosis: Involves keratinized tissues as skin, nail and hair, c. Subcutaneous mycosis: Mycosis of the subcutaneous tissues and adjacent bones d. Systemic mycosis: Starting as primary pulmonary lesions that may disseminate to any organ • An antifungal drug is the drug that selectively eliminates fungal pathogen from a host with minimal toxicity. • As fungi are eukaryotic and their ribosomes and many pathways resemble those of humans, unique fungal pathways have to be targeted Available classes of antifungal drugs: A-Interfere with cell membrane synthesis e,g Polyenes, azoles and alayllamines B- Interfere with cell wall synthesis e.g Echinocandins C-Interfere with nucleic acid synthesis e.g Flucytosine D-Miscellaneous (nucleic acid, cell membrane synthesis) e.g Grieseofulvin I-Antifungal drugs interfering with cell membrane synthesis Polyenes: fungicidal with poor gastrointestinal absorption. bind ergosterol in fungal cell membrane creating ion channels leading to leakage and cell death. Polyenes bind to less extent to cholesterol the predominant sterol in mammalian cell membranes. They are thus toxic. Amphotericin B: administered intravenously for serious systemic fungal infection caused by yeasts, molds or dimorphs but Penetration of joints and CSF is poor Nystatin: used topically to treat fungal infection in skin, vagina and oral cavity. • Azoles: The azoles are fungistatic against yeast and fungicidal against molds. Azoles inhibit P450 enzymes needed for the synthesis of ergosterol of the cell membrane and interfere with membrane integrity. They are either topically applied in skin or mucosal infections e.g miconazole or orally administered in non life threatening conditions e,g Ketoconazole 3-Triazoles: Azole drugs with three nitrogens in azole ring. They have broder spectrum and better systemic activity than azoles. Examples Fluconazole, Itraconazole, Voriconazole used in systemic infection • Allylamines: Alylamines e.g terbifunates inhibit squalene epoxidase, another enzyme required for ergosterol synthesis .This is an earlier step in the ergosterol biosynthesis than the azole class of antifungal drugs. This inhibition leads to cell membrane fragility, increased membrane permeability, and intracellular accumulation of sterol precursors. They have both fugistatic and fungicidal activity II. Antifungal drugs interfere with cell wall synthesis: 1-Echinocandins: fungicidal drugs, inhibit fungal glucan synthesis leading to weakened cell wall and cell lysis e.g caspofungin III. Antifungal drugs acting on nucleic acid Flucytosine (5-fluorocytosine): An oral fungistatic agent. It is converted in fungal cell to 5 fluorouradylic acid which competes with uracil leading to miscoding and disruption of RNA, protein and DNA synthesis. Because resistance quickly develops if used alone, it is used in combination with other antifungal drugs. It penetrates well into all tissues including cerebrospinal fluid IV. Miscellaneous: Griesofulvin; a fungistatic drug, orally administered. It is thought to inhibit fungal cell mitosis by inhibiting tubulin formation and nuclear acid synthesis. QUIZ Regarding the mode of action of antifungal drugs, which one of the following is most accurate? (A) Azole drugs, such as fluconazole, act by inhibiting ergosterol synthesis. (B) Amphotericin B acts by inhibiting fungal protein syntheses at the 40S ribosomal subunit. (C) Terbinafine acts by inhibiting fungal DNA synthesis but has no effect on DNA synthesis in human cells. (D) Echinocandins, such as caspofungin, act by inhibiting messenger RNA synthesis in yeasts but not in molds. Antifungal resistance Primary resistance: naturally among certain fungi without prior exposure to the drug. e.g. resistance of Candida krusei to fluconazole . Secondary resistance: among previously susceptible strains after exposure to the antifungal agent depends on altered gene expression (mutation) e.g. growing resistance of Candida albicans and Candida glabrata to first-line and second-line antifungal medications, namely, fluconazole and echinocandins Plasmid , transposon Drug inactivation More than one mechanism of resistance to the drug can be functioning in any given fungal strain with additive effects Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Presented by DR . Makram F. Attalah Professor of Medical Microbiology & Immunology, Faculty of Medicine, Ain Shams University 1 1 Contents • HAIs: definition, sources, predisposing factors, chain of infection • Standard Precautions • Transmission based isolation precautions • Aseptic techniques and Safe injection practices • Basics of personnel health components of occupational safety 2 Intended Learning Outcomes (ILOs) • • • • • • • • • • • • Define Health Care Associated Infections (HAI). Illustrate components of the chain of infection. Describe components of standard precautions. Apply different types of hand hygiene properly at proper situations. Identify indications for the use of personal protective equipment. Mention level of processing soiled patients care equipment. Identify types of waste in health care. Differentiate between different transmissions based isolation precautions. Describe basic principles of safe injections. Identify key processes of aseptic techniques. Recognize elements of health care personnel (HCP) safety. Demonstrate proper response to sharp injury and/ or accidental exposure to blood borne pathogens. 3 4 Healthcare associated infections (HCAIs) (Nosocomial or hospital acquired) ▪Infections acquired during health care in any health care facility ▪Not present or incubating at admission ▪Usually occurring more than 48 hours after admission 5 Chain of Infection Bacteria Virus Fungus No effective resistance to a particular pathogenic agent Patient HCP Equipment Environment Mucous membranes Broken skin Contact Droplet IPC Airborne • • • • Blood & Body fluid Respiratory droplets gastrointestinal tract: stool Genitourinary tracts: semen, vaginal secretion. 6 Endogenous (self- infection) It is caused by permanent or transient flora of the patient: • Transmission to sites outside normal habitat • Damage to tissue and impairment of primary defense mechanisms (e.g. surgical procedures) • Inappropriate antibiotic therapy (e.g. antibiotic associated diarrhea) 7 Exogenous • Other patients: either directly or through contaminated hands of healthcare personnel (cross infection). • Healthcare personnel (HCP) : if they are infected or colonized with virulent organisms • Contaminated instruments • Environment: contaminated surfaces, air or water. 8 Modes of Transmission •Contact •Droplet •Airborne 4/2021 IPC 9 Direct Contact 4/2021 IPC 10 Indirect Contact 4/2021 IPC 11 Droplet 4/2021 Air-Borne IPC 12 Predisposing Factors • Impairment of general host defense 4/2021 IPC 13 Predisposing factors (cont.) • Impairment of local host defense 4/2021 IPC 14 4/2021 IPC 15 Preventive Strategy 4/2021 IPC 16 Standard Precautions • They are a standard of care designed to reduce the risk of transmission of microorganisms from both recognized and unrecognized sources of infection in health care facilities. • They are applied to ALL patients. • They are designed to protect healthcare personnel, patients, visitors, and environment from pathogens that can be spread by: • Blood. • All body fluids, secretions, and excretions regardless of whether they contain blood (potentially infectious material). • Mucous membranes. • Non-intact skin. 4/2021 IPC 17 Standard Precautions include • Hand Hygiene • Personal Protective Equipment (PPE) (e.g. gloves, gowns, masks, eye goggles and face shields) • Proper handling and processing of patient care equipment • Environmental surfaces control • Respiratory hygiene • Safe injection 4/2021 IPC 18 4/2021 IPC 19 Hand Hygiene: a general term ➢ Hand washing ➢ Hand Antisepsis ✓ Antiseptic hand wash ✓Antiseptic hand rub ➢ Surgical hand antisepsis. 4/2021 IPC 20 Hand Antisepsis Hand Rub 4/2021 Antiseptic hand wash IPC 21 Alcohol Rub • Alcohol rub is a rapid method for hand antisepsis • It is an antiseptic NOT cleaner • Should NOT be used when hands are contaminated with visible dirt, blood or mucous 4/2021 IPC 22 Alcohol rub Easily accessible at the point-of-care Individual pocket bottles carried by all health-care workers. Bottles fixed to the patient’s bed or bedside table (or around this area). 4/2021 IPC 23 Indications of Hand Washing • Hands are visibly dirty or contaminated with proteinaceous material, or visibly soiled with blood or other body fluids, • Exposure to potential sporeforming organisms 4/2021 IPC 24 Hand rub • If hands are not visibly soiled, use an alcohol based hand rub for routine hand antisepsis The 5 Moments for HH 4/2021 IPC 25 When? The 5 moments for HH 4/2021 IPC 26 How? 4/2021 IPC 27 Steps of Hand Washing 4/2021 IPC 28 Alcohol Rub 4/2021 IPC 29 4/2021 IPC 30 Gloves Types of gloves: • Clean disposable non sterile gloves. • Surgical sterile gloves. • Utility, heavy duty gloves. 4/2021 IPC 31 Masks, eye protection, face shields Protection of HCWs • worn when there is risk of splashes or sprays of blood or body fluids into the face and eyes of health care workers (HCWs). (standard precautions) • Protection from microorganisms that are transmitted by droplets e.g. • Neisseria meningitidis, •Bordetella pertussis. (droplet precautions) 4/2021 IPC 32 Surgical standard mask Protection of patients • Trapping droplets from the wearer’s exhaled breath: to provide patient protection in the O.R. • They are worn by HCP when performing spinal procedures (e.g., lumbar punctures, myelogram, and spinal anesthesia) or inserting a Central Vascular Catheter (CVC); to protect patients from exposure to infectious agents carried in a healthcare worker’s mouth or nose. 4/2021 IPC 33 Face mask worn by the patient • Surgical mask is worn by coughing or sneezing patient to limit potential dissemination of infectious respiratory secretions from the patient to others (Respiratory Hygiene/Cough Etiquette). 4/2021 IPC 34 High Efficiency Masks (Respirator) • Designed to capture high percentages (>95%) of particles that are less than 5 micron in size • Protection of health care worker from airborne infectious agents such as Mycobacterium tuberculosis. • Also by HSCT patients when urgenly brought outside protective environment to prevent exposure to fungal spores 4/2021 IPC Respirator )N95 or FFP2( 35 Gowns and Aprons Clean Gown Sterile Gown • Protect HCWs against splashes of blood or body fluids. (standard precautions) • Asepsis in OR or inserting central lines (together with sterile gloves, standard face mask and head caps ( maximal sterile barriers) • Prevent infection transmission through contact (contact isolation precautions) 4/2021 IPC 36 Caps • To confine and contain hair during invasive procedures and inside OR 4/2021 IPC 37 Protective Foot Wear (Leg coverings, boots) Closed footwear replacing the ordinary shoes is not generally needed with the exception of some special areas such as: the Operating Room (OR) Fluid resistant closed foot wear is needed during waste management, environmental cleaning, and cleaning surgical instruments; to protect HCP from skin exposure to blood, body fluids and sharp objects like needles and syringes 4/2021 IPC 38 4/2021 IPC 39 Patient Care Equipment • Single use: proper disposal • Reusable: proper processing 2 ➢ Cleaning ➢ Disinfection ➢ Sterilization 4/2021 IPC 40 Classification of patient care equipment (Spaulding classification) •Items of high risk •Items of intermediate risk •Items of low risk 4/2021 IPC 41 Items of high risk • Penetrate sterile tissues, including body cavities and the vascular system, e.g. surgical instruments, intra-uterine devices, vascular catheters. 2 • Single use (disposable) • Reusable: Cleaning followed by sterilization 4/2021 IPC 42 Items of intermediate risk • Equipment that does not penetrate the skin or enter sterile areas of the body but is in contact with intact mucous membranes or non-intact skin • Items contaminated with virulent or transmissible organisms • Cleaning followed by disinfection 4/2021 IPC 43 Items of low risk • Items in contact with intact skin, or the inanimate environment not in contact with the patient • Noncritical patient care items: e.g. stethoscope, blood pressure cuffs • Noncritical environmental surfaces e.g. bed rails, some food utensils, bedside tables, patient furniture and floors. • Cleaning and drying is usually adequate. • Disinfection when indicated. 4/2021 IPC 44 4/2021 IPC 45 Environmental Surfaces House Keeping Surfaces Clinical Contact Surfaces • Wet Cleaning (no dry sweeping) • Spot disinfection (1/50 of 5% chlorine) • Decontamination of blood and OPIFs (1/10 of chlorine 5% Cleaning & Disinfection 4/2021 IPC 46 To sum up • HAIs are common adverse effects in all health care levels • Preventive strategy relies upon adherence to standard and transmission based isolation precautions • Aseptic techniques are basis for patient safety in invasive clinical procedures • HCP safety depends on vaccination, prevent exposure and proper management of exposure events 4/2021 IPC 47 Click to edit Master title style • Edit Master text styles • Second level • Third level Most welcome for any question • Fourth level • Fifth level 4/2021 IPC 93 Click to edit Master title style • Edit Master text styles • Second level • Third level • Fourth level • Fifth level Presented by DR . Makram F. Attalah Professor of Medical Microbiology & Immunology, Faculty of Medicine, Ain Shams University 1 Contents • HAIs: definition, sources, predisposing factors, chain of infection • Standard Precautions • Transmission based isolation precautions • Aseptic techniques and Safe injection practices • Basics of personnel health components of occupational safety 2 Intended Learning Outcomes (ILOs) • Identify types of waste in health care. • Differentiate between different transmissions based isolation precautions. • Describe basic principles of safe injections. • Identify key processes of aseptic techniques. • Recognize elements of health care personnel (HCP) safety. • Demonstrate proper response to sharp injury and/ or accidental exposure to blood borne pathogens. 3 4/2021 IPC 47 Safe waste disposal • Segregation at work place (sharp waste & infectious or regulated medical waste) • Color coding of receptacles • Puncture-resistant containers with a lid for sharp waste 4/2021 IPC 48 Infectious or regulated medical waste • Fluid waste (Blood & body fluids ). • Contaminated items with blood or other potentially infectious materials e.g. contaminated dressings. • Microbiology laboratory waste e.g. stock cultures, discarded diagnostic samples (e.g. urine, stool). • Pathological and anatomical waste. • Sharps e.g. used syringes, needles, disposable scalpels and blades. 4/2021 IPC 6 4/2021 IPC 7 Respiratory Hygiene • It applies to any person with signs of illness including cough, congestion, rhinorrhea, or increased production of respiratory secretions when entering a healthcare facility. 4/2021 IPC 8 Source Control Measures • Cover your nose and mouth with a tissue when you cough or sneeze • Throw the tissue in the trash • Wash your hands • Wear surgical mask if tolerated or maintain spatial separation, >3 feet if possible 4/2021 IPC 9 10 Safe Injection Practices • What is safe injection??? • It is a set of measures taken to perform injections in an optimally safe manner for patients, healthcare personnel, and others 11 12 Safe Injection practices • Use a sterile syringe and needle for each injection, dispose appropriately after use in approved puncture resistant container for sharps disposal. • Never use medication in a syringe for more than one patient even if the needle is changed between patients. Changing the needle but not the syringe is unacceptable. • Do not keep multi-dose vials in the immediate patient treatment area; store in accordance with the manufacture’s recommendations; discard if sterility is compromised or questionable. • Check the vial for leaks or cracks. 13 Safe Injection practices (cont.) • The vial access diaphragm should be disinfected with an approved disinfectant (70% alcohol) immediately prior to accessing. • • Always use a new needle and syringe every time fluid is withdrawn from a multidose vial. Never leave one needle inserted in the vial cap for multiple uses. • Expired medications should be discarded. 14 Proper handling of multi dose vials 1. Keep it in a clean place outside patient treatment are 2. It should be labeled with opening date and expiry date 3. Before use: Check the vial for leaks or cracks 4. Prior to access: Disinfect the vial access diaphragm with 70% alcohol 5. Always use a new needle and syringe every time fluid is withdrawn from a multidose vial 6. Never leave an inserted needle in a multidose vial 15 4/2021 IPC 16 4/2021 IPC 17 Aseptic techniques • Aseptic Technique is a general term involving practices that minimize the transmission of micro-organisms during invasive procedures • Invasive procedures • Surgical procedures • The placement of device into sterile body spaces such as intravenous lines and Urinary catheters. • Wound care • Intravenous or intramuscular injection 4/2021 IPC 62 Key processes for performing Aseptic Techniques • Hand antisepsis • Patient skin antisepsis • No-touch-technique • Suitable attire (head cap, mask, strile gown, sterile gloves) • Sterile drapes • Maintaining sterile field during surgical procedures 4/2021 IPC 63 Clean no touch technique Insertion of PVC 4/2021 IPC 21 Transmission Based Isolation Precautions 4/2021 IPC 22 Transmission based precautions •Contact precautions •Droplet precautions •Air borne precautions 4/2021 IPC 23 Contact precautions • Applied for patients infected or colonized with microorganisms transmitted by direct or indirect contact Examples : • Skin Infections: impetigo, scabies, pediculosis • Respiratory syncytial virus. • Disseminated herpes simplex, Zoster • MRSA, VRE and multi drug resistant Gram negative bacteria. • Clostridium difficile associated diseases. 4/2021 IPC 24 Requirements of Contact precautions In addition to standard precautions • Single room if available • En-suite toilet and HW basin • Clean gloves and clean gowns during any patient contact 4/2021 IPC 25 Droplet precautions Applied for patients with infections transmitted by large Droplets Examples: • Influenza • Rubella • Meningococcal meningitis • Streptococcal pharyngitis • Multi-drug resistance pneumococcal pneumonia • Pneumonic plague 4/2021 IPC 26 Requirements of droplet precautions In addition to standard precautions • Single room if available • En-suite toilet and HW basin • A standard surgical mask should be worn before entering the room or being within one meter from the patient • Limit patient movement to essential purposes • Patient can put a surgical standard mask while being outside the isolation room 4/2021 IPC 27 Airborne precautions Applied for air borne transmitted infections e.g. • Tuberculosis • Measles • Varicella-Zoster 4/2021 IPC 28 Requirements of airborne precautions • A single room with negative air flow ventilation • A high efficiency mask should be worn before entering the room. 4/2021 IPC 72 Requirements of airborne precautions • En suite toilet and a basin for hand washing. • The door of the room should be kept closed except during necessary entrances and exits. • The patient transport outside isolation room should be limited to essential purposes • Patient can put a surgical mask while being outside the isolation room 4/2021 IPC 30 Protective environment • Applied for allogeneic hematopoietic stem cell transplants (BM transplantation) • Provide engineering controls to prevent exposure to environmental fungal spores • What is the significance of protective environment? • Give reasons: HSCT patients are placed in protective environment in high risk period following transplantation? 4/2021 IPC 31 Requirements of Protective environment •What are the components of protective environment? •Positive room air pressure •HEPA filtration of incoming air •12 or more air changes per hour. 4/2021 IPC 32 Positive air pressure room 4/2021 IPC 33 4/2021 IPC 34 Risk of blood borne pathogens • Blood borne pathogens include: • HBV • HCV • HIV • They are often carried by persons unaware of their infection. • They can produce chronic infection and dangerous complications • Needle stick and other sharp injuries represent the greatest risk 4/2021 IPC 35 Safety Program • Education and Training • Immunization • Exposure Prevention • Exposure Management 4/2021 IPC 36 HCW Immunization • Hepatitis B vaccine: (3-dose vaccine series administered intramuscularly at 0, 1, and 6 months). • Influenza vaccine (annual vaccine) • Measles, Mumps and Rubella vaccines (MMR). • Varicella-zoster (VZV). • Tetanus, diphtheria and acellular pertussis(Tdap). 4/2021 IPC 37 Pre-exposure management (for HBV) • Pre-exposure evaluation for health-care personnel previously vaccinated with complete (≥3dose) HepB vaccine series who have not had postvaccination serologic testing 4/2021 IPC 38 Measure anti-HBs Antibody Level < 10 mIU/mL ≥ 10 mIU/mL Administer ONE dose of HB vaccine & re measure anti-Hbs antibodies 1 month later < 10 mIU/mL ≥ 10 mIU/mL Administer TWO dose of HB vaccine & re measure anti-Hbs antibodies 1 month after last dose < 10 mIU/mL Evaluate after every exposure ≥ 10 mIU/mL No action for prophylaxis against HBV exposure regardless of source patient Prevent sharp injuries • Be careful with sharp instruments • Do not recap by two hands • Do not dispose in plastic bags • Dispose in puncture resistant container 4/2021 IPC 41 Sharps disposal and handling √ 4/2021 √ IPC 42 Prevent mucous membrane exposure • Appropriate PPE • Use mouthpieces, resuscitation bags when needed 4/2021 IPC 43 Exposure Management • IMMEDIATE response • REPORTING • TESTING of source patient (if applicable). • Specific viruses measures 4/2021 IPC 44 Immediate Response • Skin wounds should be cleaned with soap and water. • Mucous membranes should be flushed thoroughly with water. • Eyes should be irrigated with normal saline. 4/2021 IPC 45 Immediate response to exposure incidents 4/2021 IPC 89 Importance of reporting exposure incidents (Give reasons) • Assess infection risk according to: • Type and severity of exposure: e.g. percutaneous or mucous membrane. • Type and amount fluid exposure e.g blood or body fluid. • Administer post exposure prophylaxis (PEP) if indicated. • Allow follow up for the exposed HCP. 4/2021 IPC 47 Management of exposures to specific viruses • Any blood or body fluid exposure to an unvaccinated person should lead to initiation of the hepatitis B vaccine series. • Further management of exposure to HBV depends on the HB vaccination status and the vaccine response status of the exposed personnel. • Postexposure management of exposure to HCV is intended to achieve early identification of infection by base line and follow up testing. • HCP exposed to HIV should be evaluated within hours after exposure for administration of antiretroviral medications as soon as possible after exposure within 72 hours (preferable 24 hours) 4/2021 IPC 48 To sum up • HAIs are common adverse effects in all health care levels • Preventive strategy relies upon adherence to standard and transmission based isolation precautions • Aseptic techniques are basis for patient safety in invasive clinical procedures • HCP safety depends on vaccination, prevent exposure and proper management of exposure events 4/2021 IPC 49 Click to edit Master title style • Edit Master text styles • Second level • Third level Most welcome for any question • Fourth level • Fifth level 4/2021 IPC 93
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