Lesson 1: MMI: Intro, Shapes, Gram Staining

LESSON 1: MMI: intro, shapes, gram staining 1. 2. 5 major categories Images of infection 1. Bacteriology → the study of bacteria 2. Virology → study of viruses 3. Mycology → study of fungi and yeast 4. Parasitology → study of protozoa (amoeba etc) and metazoa (worms) 5. Immunology → the study of immunity and how our body fights against infection 1. Image 2 of gangrene (black skin means the skin is dead) 1. Bacteria + infection → leg sore (image 3) 0. Virus + infection → viruses also cause infections 1. Shingles → result of the reactivation of the chicken pox virus 2. HIV → case of anal herpes (really bad infection with big areas of tissue damaged) 1. 0. Parasites + infection → microfirelia (can cause elephentatitis) 1. Leads to swollen area (accumulation of interstitial fluid) 2. Worms clog up drainage of interstitial fluid 1. 0. Immunocompromised individuals → e.g. cancer / chemo leads you to be susceptible to infection by things that do not usually cause infection 1. Bacteria and Eukarya 0. Fluid → very heavy so walking barefoot you could get a bad infection w/o even knowing abt it. (Not able to feel it) Fungus + infections 1. 0. Immunodeficiency → lack of proper treatment for HIV Ringworm and yeast infection 2. Fungal infections more severe in immunocompromised individuals than the immunocompetent 1. We will be focusing on bacteri and eukaryotic cells in this course 1. Eukarya → fungi, animals and us, amoeba, plants and etc 2. Bacteria → gram positive or gram negative Size of organisms 1. Biggest → worms (tapeworms between 7-10 meters long) 2. Bacteria → cannot be seen with the naked eye 1. 3. Have to look at them with a microscope Viruses → smaller than bacteria, have to use an electron microscope 1. 30-30,00 nanometers in size 0. D. What would happen if we didnt have bacteria in our bodies 1. We are as much bacteria as we are human 2. They provide some services to us 1. Milk contains sugars in the forms of glycans we cannot use this unless bacteria in intestines break it down 2. Production of vitamin K and vitamin B12 3. Protection against disease producing bacteria 1. If we consume something with salmonella bacteria, and we have good bacteria at receptors, bad bacteria would not be able to hook onto these receptors D. But there are some nasty bacteria 3. Imbalance can be caused by previous treatment with antibiotics 4. Infection with some strains of bacteria have virulence factors 1. 1. Naming bacteria Methods or techniques bacteria use to escape (proteins or strategies they use to avoid the WBC / immune system and cause disease 5. Immunocompromised individuals → more at risk, we need a good immune system to fight off bacteria 1. We should know genus and species and sometimes sub-species 1. 1. We would need to know genus and species 2. Classified into families based on relatedness of genetic information (use rRna for typing) 1. We need to know abbreviations so we can tell what is what. 2. 1. 2. Salmonella typhi is not a staphylococcus 1. 2. Bacterial morphology 1. 2. 3. Staphylococcus → all the species contained in the genus staphylococcus LOTS OF MEMORY WORK Cocci → round shaped 1. Staphylo → cluster of round cells 2. Strepto → chain of round cells 3. Diplo → 2 round cells together Bacilli → rod shaped 1. Diplo → 2 together 2. Strepto → a chain of bacilli 3. Cocco → a long egg shape Spiral bacteria 1. Vibrio → comma shape 2. Spirillum → big lazy filament of bacteria 3. Spirochete → long tightly coiled 1. 3. How do we tell bacteria apart if they are the same shape 1. Simple stains → used to see if bacteria is present or not, only one stain is used 1. Everything is stained the same color 1. 2. 3. Differential stains → separate bacteria into different group 1. GRAM STAIN → Divides bacteria into 1 groups 2. ACID FAST STAIN → used for mycobacterium (TB bacteria → causes tuberculosis) Most important thing to know about bacteria is the gram stain reaction 1. The gram stain and the reaction that we get is very important 1. GRAM STAINING E.g. Methylene blue Based on cell wall structure: amount of peptidoglycan (structural mesh) in the bacterial cell wall. 4. Make a slide from specimen and fix (heat or methanol) 5. Add crystal violet (primary stain) 6. Wash crystal violet off after 1 minute 1. 7. Wash off excess that does not retain on the slides Add iodine “mordant) 1. Forms a complex molecule with the crystal violet and get stuck in the mesh (both too big to get out) 8. Wash excess iodine off after 1 minute 9. Decolorization → use a mixture of acetone and alcohol 1. Drop onto surface of thre slide 2. Once the blue color leaves stop decolorization 10. Add counterstain (sarafin) → if gram negative and we cannot see the stain 11. Let slide dry and we look through the microscope 1. 2. 3. 1. Some bacteria have multiple membranes some only have a thick layer of peptidoglycan 1. Because there is no outer membrane acetone/alcohol has no effect on the outer membrane 2. Any crystal violet molecules can expect from cell so we add a counter stain 1. Stains GRAM - pink or red 2. GRAM + - purple 4. 5. Many different organisms can cause human disease 1. Summary 12. Bacteria → prokaryotic organisms (before nucleus) 13. Plants, mammals, birds, reptiles and amphibians, fungi and we humans are eukaryotes 14. Bacteria have different shapes and are named accordingly 15. Bacteria have a naming system that is dependent on their genetic relatedness The Gram stain is used to divide bacteria into 2 major groups (G+ and G-) LESSON 2: MMI, meningitis ❑ Define meningitis and explain what the meninges are SEPT 8TH LECTURE 2 - Meningitis ❑ Describe Neisseria meningitidis in terms of its Gram stain reaction and physical attributes ❑ Identify the mode of transmission for N. meningitidis ❑ List some virulence factors of N. meningitidis ❑ Identify symptoms characteristic of meningitis and sepsis ❑ Recognize the importance of a rapid diagnosis for treatment and survival 1. Neisseria meningitidis 1. Neisseria → Genus and meningitidis → the species 2. One cause of: meningitis and sepsis 1. 2. So what is meningitis 1. Many different fungi, parasites and bacteria can cause meningitis Meningitis → inflammation of the meninges 1. Adding itis → means inflammation 2. Meninges are coverings of the brain and spinal cord (protect and enclose) 3. Meningitis → usually caused by microbes infecting the cerebral spinal fluid (CSF) 1. A cushioning layer of liquid to avoid brain being knocked back into skull 4. 5. An infection does not always cause inflammation, anything that gets into the CSF can grow and reproduce and cause a lot more problems 1. Our bacterium 6. One of the microbial causes of meningitis 7. Gram negative diplococcus, has a capsule →(layer of sugars outside of the bacterial cell wall) 1. A capsule → important for disease 8. > 12 serotypes (subtypes) based on capsule carbohydrates (polysaccharides or sugars) 9. Only 6 subtypes → responsible for most of the epidemics in the world 1. A,B,C,W-135,X and Y 10. Type B → most common in developed countries 11. Only affects humans:has no animal reservoir 1. 1. Transmissio n → by droplet Cannot be something that is in animals and can infect humans 12. Sneeze, cough and saliva 13. Mucus droplets → pretty heavy 14. Range of 1-2m 2. Virulence and pathogenici ty 15. Virulence → how well the organism is able to cause disease 16. Pathogenicity → how it causes the disease 1. Attaches to epithelial cells in the throat 2. Invades and can go to the bloodstream and or CSF 3. Contains “endotoxin” anchored in the cell wall → this causes inflammation 4. Endotoxin → = (LOS) (lipo-oligopolysacchairde) has Lipid A as part of the molecule → this is the endotoxin 5. The endotoxin → causes all the inflammation that we see in the body 1. 6. Activates blood clotting, leads to DIC (disseminated intravascular coagulation), hemorrhage (bleeding into the skin), (petechiae (tiny red spots) ,purpura (big bruise like) ) and shock. Bacteria evade phagocytosis (being eaten) by WBC 2. This is possible due to the presence of a polysaccharide coating around the outside of the bacterial cell wall → the capsule 1. This allows bacteria to survive and reproduce 17. 1. Alternative names for the bacterium 2 types of disease: meningitis and sepsis 1. Sometimes called “meningococcus (sing.) (pl.meningococci)” which are nicknames for Neisseria meningitidis 18. Meningococcal meningitis ● Inflammation / infection of the meninges o Increased pressure to the brain, influx of inflammatory cells (WBC) to CSF o Death if untreated - if treated Meningococcal sepsis ● Replication of bacteria in the bloodstream o o Disseminated infection, can occur quickly, gram -, sepsis, intravascular coagulation (clotting), need for amputation Death if untreated or if treated too late early → have very good prognosis Disease → mortality = high if untreated but 90-95% chance of survival if diagnosed early and treated early. b. 0. Symptoms and disease Meningitis Sepsis ● Headache ● Fever ● Stiff neck ● Petechiae. Purpura ● Photosensitivity ● Shock ● Nausea ● Hemorrhage ● Sore throat ● DIC - internal organ meltdown ● Petechiae ● Eventual death ● Coma ● Eventual death 0. Diagnosis 0. 1. Aspirate CSF and or blood 2. Gram stain CSF and report to physician 3. Culture and or EIA / PCR 4. Serogroup for epidemiology (public health) 5. Ascertain susceptibility to antibiotics Treatment must be immediate! 1. Administer antibiotics directly after taking the culture specimens (NOT BEFORE) 2. Empirical antibiotic treatment: best guess before culture confirmation 1. 3. Best to use 3rd generation cephalosporin 1. 1. Vaccine If you suspect meningitis E.g. ceftriaxone (good CSF penetration) 4. Antibiotic susceptibility testing will be done on bacterial isolate growing from the cultures 1. 3 types 1. 2. Polysaccharide (antibodies will be made into capsule) 1. Effective against 4/15 types (A,C,Y,W-135) 2. 90% protection for 2-5 years 3. Cannot be used for pediatric patients <2 years Conjugated 1. 3. Polysacchairde combined with a protein good for children < 2 years and immunosuppressed individuals who can’t make antibodies efficiently. Serogroups A,C,W,Y Type B vaccine 1. Based on proteins in the cell wall NOT the capsule 1. New vaccine spring 2014 but varying activity in different populations 1. 2. Changing epidemiolog y Maybe 50%-60% Serogroup A used to be the most common invasive infection in Sub-Saharan Africa until 2010 when the population there were vaccinated 1. Serogroups have shifted to W,X and C in this area 0. Public Health Issues 1. Rapid ID of disease 2. Rapid ID of contacts 3. Prophylactic antibiotics 4. Vaccination of susceptible individuals 5. Health prevention information 6. Reportable disease LECTURE 3: Prokaryotic vs Eukaryotic cells LECTURE 3 : SEPT 12 Prokaryote vs Eukaryote Objectives ❑ Describe basic differences between organisms (prokaryotes vs eukaryotes) and recognize why this is important ❑ Describe basic differences between Gram negatives and Gram positives based on cell wall structure ❑ Describe the basic structures of bacterial cells and the functions of the components ❑ Identify a use for the ZN stain and know why it is used for the particular organisms ❑ Identify C. difficile as a major problem in health care, describe its characteristics and pathogenesis 1. Prokaryote versus Eukaryote 1. Prokaryote → bacteria 1. Small unicellular organisms 2. All proteins begin with a special amino acid 1. 2. Eukaryote → fungi, plant cells, protozoa, metazoa, human cells 1. All proteins begin with the amino acid 1. 3. 2. Main differences between prokaryotic and eukayotic cells Viruses, viroids and prions are not cells and are in neither category Prokaryotic cells b. b. b. b. b. b. Size 0.2-2.0µ • No nuclear membrane/nucleoli • No membrane bound organelles • Simple flagella • Cell wall complex • Plasma membrane usually lacks carbohydrates and sterols • No cytoskeleton • Smaller ribosomes (70S) • Single circular chromosome 4. • Binary fission • No meiosis 1. An enzyme → an important type of protein 2. Plasmids → contain tiny pieces of DNA 1. Eukaryotic cells 0. 10-100µ 0. • True nucleus 0. • Organelles present, e.g. Golgi, mitochondria, ER 0. • Complex flagella • Cell wall simple if present 0. • Plasma membrane contains sterols and carbohydrates 0. • Cytoskeleton 0. • Ribosomes 80S 1. • Exception: mitochondria 70S 0. • Multiple linear chromosomes 0. • Mitosis 0. • Meiosis (sexual reproduction) Adds few pieces of important games 1. Comparison of Gram negative and Gram positive organisms Mehionine 1. 1. 2. 3. 1. Formylmethionine Could be coding for antimicrobial resistance or virulence factors in exocytosis 3. Chromosomes look like spaghetti 4. Phagocytic cells do not have a nucleus 5. Gram negative: 1. Thin layer peptidoglycan 2. No teichoic acid Lipopolysaccharide 3. Outer membrane and periplasm 4. Generally less susceptible to penicillin 6. 2. Gram positive 1. Thick layer peptidoglycan 2. Teichoic acid present No lipopolysaccharide 3. No outer membrane or periplasm 4. Generally more susceptible to penicillin 2. Gram positive → dark blue/ purple stained organisms 3. Gram negative → pink/ red stained organisms Some important exceptions 1. Bacteria not stained by Gram’s stain 1. Mycobacterium sp i.e. M. tuberculosis (causes TB) 1. 2. Mycoplasma sp i.e. M. pneumoniae 2. 3. Gram stain procedure 1. Ziehl-Neelsen or “acid-fast” stain 2. 3. (cell wall has peptidoglycan but large amounts of mycolic acid which prevent stain penetration) (causes pneumonia) (no cell wall and no peptidoglycan; contains large amounts of sterols) Used for detection of Mycobacterium tuberculosis (TB) and a few other organisms with high lipid/ wax content in cell wall Few other organisms (e.g. cryptosporidium, a parasite) 1. 9. Bacterial structures and function 1. Motility 1. Flagella 2. Axial filament 1. Flagella inside cell wall of spirochaetes 3. 0. Attachment and conjugation between bacteria 1. Pili / Fimbriae Attachment to cells/tissues 2. 2. To some extent can pull bacteria along the surface of cells so motility 3. 3. Sex pilus (transfer of plasmid)e 4. Conjugation → horizontal transmission of genes from one bacteria to another 1. 5. Axial filament moves like a motor 1. 0. Screws into bacteria into tissues Capsule or glycocalyx 1. Function → evasion from WBC 2. Sometimes attachment to cells 10. Comparison of G+ and G- cell walls 11. Important cell wall structures ● Peptidoglyca:lattice strcuture like a mesh o Macromolecule, polymer of N-acetylmuramic and N-acetylglucosamine (NAG, NAMA) linked withb peptides ● Lipopolysacchairde (LPS): outer membrane (only G-) o o Lipid A: “endotoxin” anchors in bacterial outer membrane Polysacchairde = “O” antigen projects outward form cell o ● Teichoic and Lipoteichoic acids (only Gram +) o 12. Plasma membrane = cytoplasmic membrane Attached to peptidoglycan layer, project outwards and cause cells to have a negative charge ● Prokaryotes = phospholipid CM, usually no sterols in CM o ● Eukaryotes = some phospholipids, carbohydrates, sterols in CM ● Differences are important 🡪 e.g. human cell plasma membrane contains cholesterol, fungal cells plasma me contains ergosterol 13. Cytoplasm of bacteria ● Nuclear area = nucleoid: chromosome (1 in most bacteria, 46 in human cells) ● ❑ Ribosomes: site of protein synthesis, 2 subunits (70S) ● ❑ Inclusions: reserve deposits of nutrients in membrane bounded vacuoles ● ❑ Plasmids: small circular pieces of DNA that code for a restricted number of proteins 14. Endospores ● resting cells (do not metabolise nutrients): o 15. Clostridium difficile genetic material within a hard coat (dipicolinic acid and peptidoglycan), resistant to heat, drying, UV radiation, chemicals (function for bacteria is survival) ● -this is not the same as a capsule ● - Produced by only a few types of bacteria (Clostridium and Bacillus in this course) ● A major cause of nosocomial infection globally (means gotten at a hospital) ● Anaerobic Gram positive rod that produces endospores 16. What kind of organism is C.difficile ● Strict anaerobe, Gram-positive bacillus, spore forming ● ● 3-5% have in gut as normal flora 17. Virulence factors and pathogenicity ● Fecal-oral transmission ● Spores are not “living” ● Spores survive months in environment ● Spores not inactivated by antibiotics, alcohols, or alcohol based hand cleansers ● Spores are ingested, not affected by stomach acid and germinate in the intestine after contact with bile acids ● ❑ Exotoxins made by vegetative bacteria damage intestinal mucosa, kill cells and cause epithelium to leak leading to watery diarrhea (both are cytotoxins) ● ❑ TcdA or “toxin A” ● ❑ TcdB or “toxin B” ● ❑ CDT – C. difficile binary toxin: often but not always found in epidemic strains An emerging problem in hospitals: ❑ NAP1/B1/027 - new more virulent strain 16X more toxin, 3-4 X mortality rate, faster sporulation, high resistance to fluoroquinolone antibiotics, mutation in tcdC which is the toxin regulator gene 18. What syndromes are common with this bacterium? ● Pseudomembranous colitis ● 19. Treatment of Clostridium difficle ● Conventional 🡪 antibiotics like vancomycin and metronidazole ● Nonconventional 🡪 probiotics, fecal implants (best success rate of all treatments) MMI 133: Lecture 4 Bacterial Growth, enzymes, metabolism, phases of growth SEPT 14: Bacterial Growth Objectives Endotoxin: e.g. Lipid A in LPS Describe how bacterial metabolism has an effect on rate of growth ❑ Explain the effect of discussed growth requirements of bacteria (ie nutrient, gaseous, pH, temp) ❑ Identify Listeria monocytogenes as a problem for food borne infections and describe its characteristics and pathogenesis ❑ Define the kinetics and terminology used for bacterial growth ❑ Define and describe differential versus selective media for bacterial growth. Part of the cell structure-anchored in outer membrane – Lipid molecule (Fat) – Released from the cell when the cell dies and disintegrates ● The endotoxin LIPID A is the cause of all the bad things that we can see in certain diseases e.g. meningitis ● These are part of the cell wall structure and are not released unless the cell dies Exotoxin: e.g. tetanus toxin, botulinum Bacterial Metabolism and Growth Produced by the bacterial cell (ribosomes synthesize); not a part of the cell structure – Released from cell in many ways, damages other cells – Protein – Three functional types: enterotoxin, neurotoxin and cytotoxin ● Metabolism: sum total of all chemical reactions within an organism ● catabolic: release energy ▪ In catabolic reactions we release energy to break down sugars ● - anabolic: store energy ▪ In anabolic reactions we store energy in large amounts to break down later ● Catabolism: chemical reactions that release energy, breakdown of organic compound Anabolism: - chemical reactions that require energy, building of complex organic molecules from simpler compounds (stores ATP) . i.e. formation of polysaccharides from simple sugars like glycogen from glucose ● Glycogen is released / broken down to produce energy from glucose when we need it Reaction Rates: ● temperature dependent, higher the temp, higher the reaction rate, but…. ● drawback: high temperature may kill cells (denaturation of proteins) ● A solution to this problem is enzymes 🡪 these are proteins deteriorate at higher temperatures than their usual Enzymes ● Large protein molecules (biological catalysts) ● Make chemical reactions happen 100,000,000 X faster ● Substrate specific (what they work on) o Whatever the substrate is ● Have suffix “ase” ● May need cofactors – e.g. metal ions like zinc, magnesium ● Recyclable, unchanged during reaction Enzymes: function can be affected by cellular controls Temperature pH Saturation Salt concentration Inhibitors, i.e. mercury, silver Energy production most microbes use carbohydrates as 1 source for production of energy  glucose the most common nutrient source Two processes by which glucose is used: a. cellular respiration aerobic anaerobic b. fermentation -both processes use glycolysis (e.g. Embden-Meyerhof pathway) where the final product is pyruvate Bottom Line: Need to produce ATP (energy) to run cell processes Respiration: Aerobic and Anaerobic Aerobic Respiration: -produces more ATP than anaerobic processes, so these “aerobes” usually can grow faster (~38 ATP from 1 glucose molecule) -bacteria that need oxygen in order to grow are called obligate aerobes Anaerobic Respiration: - less ATP is produced (2-4 ATP) -bacteria usually grow slower - bacteria that can only grow without oxygen are called obligate anaerobes Energy sources other than glucose: Lipids (fats): Lipids and Protein -broken down by lipases: fatty acids and glycerol which enter the Krebs cycle  Proteins -broken down by proteases, end up in Krebs cycle Factors affecting bacterial growth Physical Factors 1. Temperature 2. pH 3. Osmotic pressure 4. Gas B. Chemical Factors -Metals, nutrients, etc C. Environmental Factors -Intracellular growth Physical factors 1. 2. 3. 4. 5. Temperature 1. Psychrophiles 🡪 (-5 to + 15 degree Celsius) Psychtrohtrophs 🡪 (20-30) (e.g. listeria) Mesophiles 🡪 (25-45) 1. Most pathogens are in this group Thermophiles 🡪 (45-70) Hyperthermophiles 🡪 (70-110) Bacterial growth contd ● pH (5-8 usual aactivity zone for human pathogens 🡪 neutrophiles ● If the pH is too high or low then the functions deteriorates o o The protein will begin to unravel and cannot function properly / the way it was meant to The nooks and crannies will no longer exist Listeria Monocytogenes Gram positive bacillus, nonspore forming, psychrotroph ❑ facultatively anaerobic, motile ● Can grow with or without oxygen (facultative) ❑ grow well on complex media (BAP – blood agar plate) ❑ grow over wide temp. range (342 °C) ● Makes it the perfect foodborne pathogen ❑ grow over wide pH range (5.5 9.5) ● Can grow regardless of where it is ❑ grow in high concentrations of NaCl (up to 10%) ● Can even grow in huge concentrations of salt Listeria Growth ● Normal habitat o o o Clinal presentation of Listeriosis Widely distributed in nature, soil 🡪 natural reservoir Animal intestine through contaminated feed Hardy 🡪 survives in low temps, high acidity and salt concentrations ● Cause disease in man and animals ● In animals: o CNS infection, goats, cattle 🡪 circling disease ● In man: o o o Virulence and pathogenesis (how it causes disease) Food borne disease 🡪 more likely in immunocompetent individuals CNS infections. Death Abortion 🡪 miscarriage in pregnant women ● Ingestion of contaminated food ● -invasion of bacteria through gastric epithelial cells (rearrangement of actin propels bacteria from one cell through the next cell membrane into another cell like a rocket) (gene ActA codes for this protein- an “invasin”) 🡪 invasin = virulence factor that helps bacteria invade ● grows in macrophages, destroys the phagolysosome (membrane bound compartment that the bacteria are taken into the cell in) with listerolysin ● produces two other hemolysins (phospholipases) that destroy cell membranes 🡪 (this bacteria can easily escape phospholysosomes) ● Intracellular and extracellular growth 🡪 can grow outside or inside of the host cell ● Never goes to the outside where WBC are Clinical infection ● Meningitis, encephalitis, septicemia o o o The incubation period 🡪 as long as 2 months (hard to tell what you ate 2 months ago) The elderly and immunocompromised are at greater risk Pregnant women also at risk 🡪 they get influenza like bacteremia illness that can result in abortion or still birth Transmission ● Fecal-oral ● Foods implicated as a vehicle of infection: o Epidemiology coleslaw soft unpasteurized cheeses turkey wieners, cold cuts mushrooms and prepackaged salads milk ● Hard to track because of long incubation period and underreporting o The period in which you get infected and the time it takes for the symptoms to first present ● Estimated to be responsible for 1700 cases of invasive disease in the US each year ● Fatal in about 1/3 of the cases even with appropriate antibiotic treatment ● Maple lead incident 🡪 2008: 57 severe infections: 23 deaths Prevention and treatment ● No vaccine ● Antibiotics for treatment but you have to diagnose the infection in time Osmotic pressure: nature wants to balance ● Isotonic solution 🡪 e.g. 0.85% NaCl o This is considered the normal amount of saline water ● Hypotonic solution: fewer particles/ml in solution than in the inside of a cell. Water then enters the cell to even the osmolality (concentration of particles/ml) – and causes the cell to expand and explode o The cell will blow up and eventually explode o Water enters the cells ● Hypertonic solution: more particles/ml in solution than in the cell. Water leaves the inside of the cell causing the cell to dehydrate, cell membrane to collapse. This is plasmolysis (causes growth inhibition) o In this case water leaves the cell and it will shrivel up ● Within the food industry this is used to kill bacteria 🡪 for example salt is used for pickling Osmotic pressure ● Some bacteria have developed a tolerance to hypertonic environments ● Obligate halophiles 🡪 need high salt concentrations ● Dead sea halophiles 🡪 need 30% salt to grow (these are not human pathogens) ● Facultative halophiles tolerate some levels of salt 🡪 2%-15% o Enterococcus specieis, vibrio specieis ( enteric bacteria 🡪 bacteria in intestines), some staphylococci (usually on skin) o Gaseous requirements for growth ● Oxygen requirement can be used to classify bacteria 1. 2. 3. Strict aerobe 🡪 need oxygen to grow Facultative anaerobe 🡪 grow at the interface but also grow in places without oxyegn Strict anaerobe 🡪 only growth without oxgen so grow at the bottom Aerotolerant anaerobe 🡪 Produce enzymes that break down oxyegn so grow all over Microaerophile 🡪 Only like oxygen at the concentratuion of what we have in air, do not go higher or lower 4. 5. Chemical factors affecting bacterial growth ● Sources of C,N,O,S,P and trace elements ● Sources of organic growht factors o o Intracellular growth E.g. yeast extract in artifical media E.g. cysteine or other amino acids ● Intracellular organisms o o o o evade defense mechanisms (e.g. WBC) need for antibiotics that penetrate host cell membranes in order to treat infection some cannot be grown on artificial media (must be cultured in living cell culture) may be dependent on host cell for energy (i.e. Chlamydia which lack enzymes for ATP production) ● Bacterial Growth 1. 2. 3. 4. Reproducition 1. Bacterual reproduction 🡪 binary fiission 2. Generation time 1. Time for one bacterium to divide into 2 2. Dependent on media and growth conditions 3. Aerobic bacteria usually grow much faster than anaerobic bacteria ( increase in production of ATP) 1. Do not need to memrozie these generation times 2. We use logarithmic expression 🡪 to express bacterial number Grwoth kinetics 1. Log phase -🡪 grows the most, most exponential growth 2. Biofilms 1. Bacteria like to live in communities 2. >85% of human infection involves biofilms 3. Dynamic 🡪 bacteria are not stuck, they can leave the biofilm and start a new focus of infection 4. Slimy matrix with polysaachrides, proteins, DNA, bacteria 5. Antibiotics = ineffective against the bactria living in bioflims 1. Cannot penetrate 5. Bacterial cultures 2. Reality Micro: how big they grow in the lab 1. need nutrient material (solid, semi-solid or liquid) 2. nutrient medium should be sterile to start with! 3. -agar: complex polysaccharide from algae for solid culture matrixlooks like gelatin 4. -often add animal blood (sheep, horse) for nutrients 5. -bacteria grow in “colonies” – piles of bacteria where the original bacteria was 1. placed on the surface of the agar filled plate (petri dish) ● Often use broth or semi-solid growth media (agar) with added nutrients and or selective agents (chemicals or antimicorbials) ● We have to know the grwoth requirements in order to successfully cultivate the bacteria we are interested in o e.g. Haemophilus influenzae needs hemin ( from blood) and o NAD (from yeast extract) ▪ Legionella pneumophila needs L-cysteine to grow ● “complex” media is ogten used for grwoing most human pathogens o Hemolysis (e.g. SBA (sheep bood agar), chocolate agar – same as SBA but blood has been heated and denatured (turns brown) ● Alpha α: partial breakdown of RBC (greening) around & under colony ● Beta ß: total breakdown (clearing) of RBC around & under colony ● Gamma γ: no breakdown of RBCs around or under colony Differential media ● Ingredients in media allow us to tentatively identify bacteria, at least into major groups (most bacteria will grow) o Blood agar for Streptococcus (hemolysis) Example of a combined selective and differential agar medium ● MacConkey plate 🡪 inhibits growth of Gram positives and contains lactose to differentiate between lactose fermenting and non-fermenting Gram negatives. MMI Lecture 5: Viruses Objectives for session 5 Virus analogy Differentiate between bacteria, virus and prions ❑ Describe the general structure of enveloped and naked viruses ❑ Understand how viruses replicate using a cell ❑ Explain how a virus can be a cause of cancer ❑ Discriminate between active, latent and chronic infections with virus ❑ Describe the disease “chickenpox”and the reactivation “shingles”- differentiate from “smallpox” ❑ Understand the nature of prions and give examples of prion diseases ● Spaceship without fuel tanks (energy reserves) ● Destined to drift in space until it can dock with the appropriate “mother-ship” ( host cell) and offload cargo ● Cargo = viral genome takes over the host cell machinery and produces many “clones” (new viruses) o The genome or genetic material ● New viruses leave the host cell and drift, looking for their own host cells to infect, sometimes take a souvenir coat (the coat is within the membrane) when they leave the host cell o Naked viruses are hard to defend against, antibiotics do not respond well o Coated viruses are easier to get rid of ● Infectious particles, but not living cells Viruses ● Responsible for majority of visits to family physician ● Many ways they can be transmitted from host to host o o E.g. blood transfusion, mucus droplets, (these droplets 🡪2m) t ▪ o o aerosols, fomites, ▪ o o This also includes sprays of droplets, and grocery water Found mostly on inanimate objects 🡪 door knobs, water bottles, counters and etc. air (airborne), water, ▪ Mucus droplets, water/air/particles in the air that bacteria can hitchhike off of. ▪ o o Can stay in the air for 4hours+ food, vectors ● Must be treated using different therapeutic strategies than other microorganisms because they are different in make up and structure Viruses vs Bacteria Virus Smaller: 10-500 nm Bacteria Larger: 500 - ~2000 nm ❑ Most are free-living and multiply outside of host cells ❑ Multiply only in host cells ❑ Contain DNA or RNA, never both ❑ Few enzymes ● Usually only the enzymes that are ❑ Always contain DNA and RNA ❑ Contain many enzymes (metabolic ones) ❑ Contain ribosomes and enzymes for metabolism of nutrients needed to start the process ❑ No ribosomes or enzymes for metabolizing nutrients Virus Sizes and shapes ● Viruses are smaller than most bacteria ● They are always obligate intercellular organisms ● Virion 🡪 complete infectious viral paerticle with nucleic acid surrounded by a Viral structure protein coat o RNA or DNA (ss 🡪 single, ds🡪 double) + “capsid” (protein coat) compromised of “capsomeres” (protein molecules ● Complete unit: nucleocapsid ● Some viruses have “lipid envelopes” outside of the nucleocapsid (protein layer) o Envelopes are derived from host cell membranes: ▪ o E.g. influenza virus, herpers virus, HBV, HIV Glycoproteins or spikes inserted in envelopes or capsid ▪ Glycoproteins 🡪 attached in protein later of naked virus Influenza virus ● Neuraminidase 🡪 when a cell is infected with a lot of these help transfer the virus to other cells ● Hemagglutinin 🡪 Major glycoprotein: helps bind to cell surfaces of virus + mediates fusion of virus into other cells. Examples of Viral names ● Herpes simplex type 1 (HSV-1) o Cold sores 🡪 can cause genital herpes w oral -genital contact ● Herpes simplex type 2 (HSV-2) o Genital sores 🡪 can cause oral cold sores ● Cytomegalovirus (CMV) o Named for enlarging cell with growth – HHV 5 ● Smallpox (Variola major) o Or variola minor ● Naming is not the same as with bacteria Host range ● Different viruses can infect every form o Bacteria (only), fungi (only), plants (only), animals (only) and humans ▪ Although some animal viruses can affect humans, but humans cannot be affected by plant viruses ● Usually fairly specific “ host range or specifcitiy) o Bacteriophages E.G. bacteriophage infects only bacteria ● This is another way to transmit viruses ● Viruses that infect bacterial cells and can transfer new genes from one bacteria to another ● Can be used as a tool for genetic engineering o E.g. ptoduction of human insulin from E.coli ● Can transfer genes for production of toxin o o Cliassifying viruses Corynebacterium diphtheriae (lysogeny) 🡪 Bacteriophage replicates their DNA using that hosts DNA ● Viruses are grouped into families on basis of DNA or RNA genome composition o o o E.g. Herpeesviruses are DNA viruses Influenza A, Measles, Rubella, HIV are RNA RNA viruses are known for their ability to mutate quickly ▪ No proof reading aspects in RNA leading to more replication + more genome mutation (if gene code is incorrect, nothing will stop it from mutating) DNA viruses (6) Papillomaviridae (HPV) ❑ Adenoviridae (adenovirus) ❑ Hepadnaviridae (hepatitis B virus) ❑ Herpesviridae (HSV-1, HSV-2, HHV-3….) ❑ Poxviridae (smallpox, monkeypox etc) ❑ Parvoviridae (B-19) RNA viruses RNA viruses ❑ Retroviridae (HIV) ❑ Flaviviridae (Yellow fever virus, West Nile virus) ❑ Orthomyxoviridae (Influenza A,B,C) ❑ Picornoviridae (Enterovirus) ❑ Coronavirus etc… Events occurring in viral infection 1. 2. Attachment and penetration into host cell 1. Virus must attach to the host cell, usually to receptors 2. Two processes for penetration of the virus into the host cell Fusion 🡪 for enveloped viruses 1. 1. 3. Virus fuses with cell membrane, becomes one with cell, virus drops material into cell Pinocytosis 🡪 for naked viruses 1. 1. 3. Replication of the genome 1. DNA forms the genome of all organisms except for some viruses and prions (which do not have a genome). The sequence of the nucleotides that make up the DNA (A,T,C,G) will determine the genes present. 2. Important reminders Uses the cell membrane to envelope the virus 🡪 virus is combined in a vacuole 🡪 vacuole has to be disrupted to implant virus material Transcription of the DNA gives rise to a RNA molecule that is almost an exact copy of the DNA. mRNA (messenger RNA) leaves the nucleus and goes to the ribosomes where the amino acids are assembled according to the instructions given by the mRNA – then the protein is synthesized. ● Remember that viruses have only one type of nucleic acid (DNA or RNA) ● The goal of a virus is to replicate itself o o A DNA virus often hijacks the host cell DNA polymerase to produce viral DNA A RNA virus must bring its own enzyme into the host cell because it needs to produce RNA from RNA to make a new virus ● We lack the enzyme: we can make RNA from DNA but not DNA from RNA or RNA from RNA DNA vs RNA vs Retroviruses viruses ● ● ● The replication of viruses 1. 2. 3. Assembly and Release of New Virus DNA virus: host cell DNA polymerase may be used directly to make more virus DNA (if replic in host cell nucleus) 1. Vrial DNA 🡪 viral DNA RNA virus: virus must carry its own RNA polymerase enzyme to producd RNA from viral RNA (no RNA polymerase in host cells) 1. Viral RNA 🡪 viral RNA RNA retrovirus (i.e HIV) carries its own reverse transcriptase enzyme in the virus capsid: 1. Viral RNA 🡪 viral DNA integrated into the chromosome 🡪 viral RNA ● Association of newly replicated RNA or DNA (nucleic acid) with newly made viral proteins results in new nucleocapsids ● If enveloped virus: envelope components are produced and inserted into the host cell plasma membrane, the viral particle then attaches to the plasma membrane and buds through 1. 2. Antiviral drugs target events occurring in viral infection 3. Host-virus infections Fuzeon 🡪 stops entry (fusion) of HIV into cells Acyclovir 🡪 stops replication of herpes viruses by interfering with the production of the viral DNA polymerase TamiFlu (oseltamivir) 🡪 stops budding/ release of influenza virus from host cell (keeps from busting) ● Acute or productive infections (feel symptoms, lousy, sick) ● Virus replicates, produces many virions. ● Host cell often killed = lytic infection (adenovirus, poliovirus) ● Latent infection (for life) ● Viral genome persist in host cell but does not replicate ● (provirus) (herpesviruses, HIV) ● Chronic infection ● Virus replicates without causing host cell lysis (death) and can persist for long periods of time without symptoms (hepatitis C virus, hepatitis B virus) 🡪 only noticeable until overabundance of liver damage 🡪 then symptoms Acute chicken pox infection ● Caused by an enveloped DNA virus, HHV-3 or human herpesvirus -3, sometimes called varicella Acute infection that becomes latent ● Acute infection ● Fever, itchy (pruritic) rash (5 days) ● Rash is spread on trunk and head – less on arms and legs ● Virus particles in rash can escape and infect – classified as an airborne virus ● Skin vesicles always present in different stages of lesion formation ● When lesions heal, the virus travels up the nerve that enervates the skin at the sire of the lesion and makes a home in the peripheral nerve ganglion where it remains latent (forever) ● Can reactivate as shingles (crawl back up nerves) Chickenpox: acute and latent infections ● Latent infection ● Virus is present but is not replicating until triggered by external factors, then travels down nerve to skin and reactivates ● In the case of Human Herpes virus 3 🡪 reactivation known as shingles or zoster ● Triggers? Age? Decline in immune system? ● Shingles or Zoster (reactivation of latent infection) Stress, UV radiation ● Reactivation of virus as it travels back down the nerve to the skin from the site of latency ● Begins as a local skin rash ● Characterized by confluent rash and pain ● Untreated rash lasts 205 weeks, infectious to those who have not ha chickenpox ChickenpoxL complications ● Most common complication in immunocompromised is secondary bacterial infection (when children scratch the lesions) ● Chickenpox parties bad idea! (increases change for flesh eating disease in children) ● CNS disease 🡪 encephalitis, meningitis, myelitis Zoster: complications ● Hemorrhagic complications (rare) ● Primary varicella pneumonia very rare – but very serious ● PHN (Post Herpetic Neuralgia) most common complication ❑ 25-50% of patients >50 yrs develop PHN ❑ Pain that persists for months or years ❑ Can cause permanent nerve damage so important to get treatment (acyclovir) within 48-72 hours of symptoms appearing Immunocompetent Zoster Immunosuppressed zoster Prevention of chickenpox (HHV-3) ● Active immunization: ● Live attenuated vaccine, highly protective, memory cells produced ● Now can be included with MMR (MMRV) ● Passive immunization ● Effectiveup to 3 days post-exposure ● VZIG: varicella-zoster immunoglobulin (antibodies to the virus) 🡪 that were produced in healthy individual ● Normally used for immunocompromised exposed children and neonates born to mothers with varicella Chicken pox vs smallpox ● Chicken pox 🡪 lesions are in different stages of development ● Smallpox 🡪 all lesions are in the same stage of development (more about smallpox in lecture 13) Oncogenic viruses ● Some viruses can transform norml host cells to cancer cells ● Not all transformed cells become cancerous e.g. wart virus often becomes a benign tumour ● Cancer-producing viruses can be RNA or DNA viruses Burkitt’s Lymphoma is associated with a herpesvirus. Epstein-Barr virus ● HHV-5 Viral diagnosis: PCR (common) or cell culture (less common, older method) ● Culture: patient specimens added to cultured cells in the lab and after a period for growth, observed for CPE (cytopathic effects) on the cultured cells (virus will cause characteristic changes in cells they infect) ● Labour intensive, slow (dependent on viral growth rates_ ● PCR = polymerase chain reaction ● Molecular biology technique used to detect the genes in an organism Syncytia (a type of CPE) 🡪 cytopathic effect Prions ● Defined as late as 1982 by Stanley Prusiner ● Proteins misfolding disease 🡪 produced as long chain ● Self protein becomes changed and non-functional ● Prions are misfolded proteins that act as infectious agents in susceptible exposed animals ● Examples of prion disease: ● Mad Cow Disease (BSE, bovine spongiform encephalitis) Scrapie in sheep Kuru in N. Guinea (transmitted by cannibalism) CJD, vCJD in humans 🡪 may be bovine Fatal familial insomnia 🡪 potentially from genetics Protein folding ● Human prion disease ● Always fatal o Signs: ▪ Ataxia ▪ Cortical visual symptoms ▪ Progressive dementia ▪ Myoclonus ▪ Akinetic mutism ● Average time to death after onset 🡪 4 months MMI 133 Lecture 6: parasites Objectives: ● Differentiate between protozoa and metazoan ● Escribe how the different parasitic organisms cause disease in humans ● Identify the organisms causing the specific diseases and identity pathogenic characteristics (i.e Plasmodium falciparum) ● Identify some commonly occurring ecto-parasitic infections (infections caused by lice / mice, live on the surface ● Describe the epidemiology and transmission of parasites ● Protozoa 🡪 once celled eukaryotes Parasites ● Metazoa 🡪 multicellular animals (Helminths) ● Ectoparasites 🡪 free living “bugs” ● Protozoa Some have a “cyst” form with a tough protective layer outside of the cytoplasmic membrane – this gives protection from inhospitable environments ❑ Growing form in many species called “trophozoite” – there is no cyst wall just a cytoplasmic membrane ● If they leave the body and enter the water. If they did not form a cyst it will blow up like a balloon and die Protozoans to know ❑ Cyst form may also be called an “oocyst” in some types of protozoa e.g. Cryptosporidium ● Cryptosporidium 🡪 causes diarrhea that can be chronic (form in the oocyst) ● Entamoeba histolytica ● Giardia lamblia ● Trichomonas vaginalis ● Acanthamoeba ● Cryptosporidium Toxoplasma gondii ● Trypanosoma ● Leishmania ● Plasmodium ● Most cause gastrointestinal diseases and the last 3 are tropical Entomoeba histolytica ● Agent of amoebic dysterney ● Primary good → RBC causes ulcer formation and bloody diarrhea ● Both trophozoite and cyst form (semi dormant and form we drink in water) ● Cyst has a high resistance to chlorine ● Can be treated with a nitroimidazole → antibiotic that treats anaerobic bacteria and metronidazole ( Flagyl) ● Cyst in fecal material → usually only see 4 nuclei in these cysts ● When the cyst wall leaves protozoa will look like green image, they like to eat RBC ● Also likes to produce flask shaped ulcers Giardia lamblia ● Flagellate: causative agent of giardiasis, beaver fever ● Intestinal upsets, flatulence, nausea, abdominal cramps and diarrhea ● Cyst → hardy, high resistance to chlorine ● Can be treated with nitroimidazoles Trichomonas vaginalis “Trich” ● Flagellate, motile ● No cyst stage, person to person infection - transmission sexual ● Found in vagina and male urethra ● Intense itching, inflammation of tissues ● Can be treated with nitroimidazoles Acanthamoeba sp ● Grows in water, exists as cyst in dust ● Important for contact lense wearers ● Eye loss outcome of infection ● Cyst trophozoite stage in humans ● Difficult to treat Cryptosporidium parvum ● Cows, rats, dogs, cats, to people ● Chronic diarrhea severe in immunocompromised individuals ● Diarrhea is serious even in immunocompetent ● Must use ZN stain ● Oocysts have high resistance to chlorine ● No good treatment Toxoplasma gondiii ● Life cycle in cats ● Cat feces contain oocysts ● Oocysts ingested by human or animals ● Tissue cyst containing the protozoan can form in other animals ● Acquired from undercooked meat (tissue cysts) or from contact with cat feces (oocysts) ● Serious for fetus in pregnancy o o o Especially first trimester Pregnant women should eat well done meat and not change the litter box Pregnant women in danger → can get into blood supply and get to the fetus Trypanosoma ● Blood parasites, transmission mostly vectros (bugs) ● Two types → Trypanosoma o T. brucei gambiense/ rhodesiense ▪ o o T.cruzi causes Chaga’s Disease in South America (reduviid or kissing bug = vector) ▪ Leishmania sp. Causes african sleeping sickness (Africa-tsetse fly → vector) Chaga’s disease, very common in SA - 10% cardiac mortality ● Leishmania species o 20 different species cause disease, all transmitted by sandfly vector o Visceral, cutaneous and mucocutaneous syndromes o Live and develop in macrophages (immune cells) Plasmodium species ● Cause of malaria, no vaccine “apicomplxa” parasites ● About 2-4 million deaths a year mostly children ● 4 main types → all transmitted by Anopheles musquito o P. falciarum o P. malariae o P. ovale o P. vivax o New species → P.knowlesii (called monkey malaria) ● Most dangerous type → p.falciparum (infects all ages of RBC → high level of parasitized RBCs) ● Mosquito carries the sporozoite in saliva, this enters the bloodstream, then liver cells within 30min ● Focus of infection in liver Symptoms of falciparum malaria ● Fever and chills caused by releae of toxic breakdown products when the RBCs rupture and release developing parasites ● Sequestration (sticky collections) of parasites and red blood cells occlude capillaries causing organ death. This is due to a virulence factor called PfeMP1 (a protein) ● Severe consequences of P.falciparum malaria → o Cerebral malaria → clumping and occlusion of blood vessels, also vasculitis o Anemia → caused by lysis of RBC o Kidney failure → caused by high levels of hemoglobin breakdown products. ● Worms → metazoa 1. Nematode 1. Intestinal and blood nematodes 2. Trematodes 3. Cestodes 0. Nematodes 1. Roundworms with complete digestive system including mouth, intestine and anus 2. Two categories 1. Eggs are the infective stage 1. Enterobius vermicularis (pinworm) 1. Live in large intestine 2. Females migrate to anus, lay eggs on skin around anus 2. Produces a glue (irritant) to stick to eggs to the skin 3. Usually not serious 4. Small size, females 1cm 5. Eggs are the infectious stage and transmitted by direct contact with eggs or ingestion of eggs stirred up in dust 1. Ascaris lumbricoides Very infectious → day care settings and etc ● Large round worms, about 30cm long ● Lives in small intestine of pigs, cows, horses ● Eggs in soil → very hardy (thick protein coat around egg) ● Infection by eggs ● Adult worms distinct sexes (dioecious) ● Diagnosis of infection by examination of feces ● Ascaris eggs hatch intestine, the larvae → tracheae → nose or mouth (children) o In children → more common since they lack swallow reflex 0. 1. b. b. Trichinella spiralis Larva are infective stage Hookworms 1. Infection by larvae, enter intact skin of bare feet 2. Lymph and circulatory system → lungs → coughed up and swallowed down to the small intestine, where attached by hooks on the scolex (head) 3. Suck blood →cause anemia 4. Eggs excreted 5. Only found in warm climates 2. Anisakis A cod worm 1. Fish nematodes, accidentally infect humans in larval form 1. Rare type pf infection, but increasing in prevalence 1. Prevention 1. Heat to 60 for 10 min or blast-freeze to -35 for q5 hours or freeze at -23 for 7 days 1. Cleaning of fish (evisceration) as soon as possible ● Ingestion of encysted larvae in undercooked, pork, boar, bear, walrus ● Larvae encyst in striated muscle fibers (and heart) ● Muscle weakness is dominant symptom ● Difficult to treat ● Main filarial infections are o Lymphatic filariasis (elephantitis) ▪ o Onchocerciasis (river blindness) ▪ o Adult worms live in subcutaneous tissue Microfilariae (offspring of adult worms) ▪ o Adult worms live in lymph tissue Circulate through bloodstream or migrate through subcutaneous tissue Transmitted by mosquitoes ▪ High prevalence in Africa, Asia and tropical Latin America Arthropods that carry pathogenic microorganisms are called ● Vectors Trematodes (Flukes) (leaf shaped) Schistosoma ● Human is infected by cercariae (larval forms) that penetrate intact skin in fresh water ponds ● Adult worms hide from host immune system under a coat of protein acquired from the host ● Worms reside :n the venous plexus of bladder or intestine, depending on the species ● Eggs cause inflammation (not adult worms) 2 types of :schistosoma in humans ● Swimmers itch ● Caused by a duck schistosome ● These cercariae do not enter the human circulation, self limiting, causes skin itching due to immune system Cestodes (tapeworm) ● Intestinal parasites, dependent on host for nutrients, no digestive system ● Head or (scolex) has suckers or hooks for attachment to gastric mucosa ● Body consists of segments called proglottids, each of which contains male and female reproductive organs ● ● Acquired by eating inadequately cooked contaminated beef, pork or fish ● Intestinal infection: milf clinical picture, produced by pork, beef, fish or rodent tapeworms ● Deep tissue infection: serious infections, pork tapeworm (cysticercosis) or echinococcosis (dog tapeworm) ● o Can end up in the brain ans damage the brain tissue Taenia saginata: beef tapeworm ● Life cycle requires both humans and cattle ● Infectious tissue larvae (cycteric) ingested by humans in infested meat, mature into adult tapeworms in intestine ● Adult worms may be 10m long ● Eggs passed in feces, may be taken up by cattle ● Good for weight loss? Taeia solium: pork tapeworm ● More dangerous type of tapeworm o Same life cycle as beef tapeworm but intermediate host is the pig cell o More dangerous than beef tapeworm (cerebral cysts cause cysticercosis), person to person transmission is possible o 2 modes of infection ▪ Eggs → result in hatching of larva in intestine, which then escape and penetrate body tissues, including brain ▪ ▪ Encyst in brain and cause fainting and seizures. Larvae → result in adult worm in intestine Echinococcus granulosus (dog tapeworm) ● Small tapeworm, humans are dead end hosts ● Dogs, coyotes, dingos ● Larva develops into a hydatid cyst (a bubble with different heads and hooks) with hundreds of worm parts but cannot form a whole tapeworm in human Ectoparasites ● Live on skin, do not enter deep tissues ● Often result in secondary infections due to scratching ● Scabies ● Sarcoptes scabei o Finger webs, elbows, armpit, breasts, groin and buttocks, rarely on head o Mites burrow into skin, feed, life span = 30 days → mate, lay eggs in burrow o Host defenses cause inflammation around the burrows, intestine itching o Secondary infections with skin bacteria o Other sarcoptes mites ▪ Dog mange, do not thrive on humans and do not establish infection ● Bed bugs → also ectoparasites o Pubice lice or “Crabs” (Phthirus pubis) and had lice (pediculus humanus) Not dangerous but itch a ton ● Surface dwellers ● Penetrate skin with mouth, suck blood ● Female glues eggs to hair shafts ● Eggs hatch in 5-10 days ● Secondary infections when lice feces are crushed into wounds ● Can also attach to eyebrows and eyelashes Summary ● Two general types of parasites (protozoa + metazoa) (helminths) ● Protozoa of importance for diarrhea even in Canada ● Other protozoa transmitted by vectors and infect internal organs, CNS etc (e.g. Trypanosoma malaria) ● Helminths (worms): nematodes, trematodes and cestodes (usually no diarrhea) ● Filaria are nematodes MMI 133: Lecture 7, Fungi and Yeast ● Differentiate fungi from bacteria, viruses and parasites Objectives ● Identify structures of fungi that can be used as targets for antifungal drugs ● Describe the characteristics of “dimorphic fungi” ● Define 3 types of fungal infection: superficial, cutaneous and systemic) and name a common dermatophytic diseases ● Define opportunistic, and give examples of opportunistic fungal infections Fungi ● Important as nosocomial (hospital acquired) infections and as opportunistic infections of immunocompromised, a few are primary pathogens ● Many beneficial functions in nature o Decomposition of plant matter, edible mushrooms ● Leavening agent for bread, fermentation for beer, wine and etc Ergosterol – our target! ● cell walls of fungi o Chitin - stains with calcium binding agents - Calcoflour white - for rapid identification fungi in clinical specimens ▪ o Ergosterol in cell membrane (we have cholesterol) Many antifungal drugs affect ergosterol synthesis. ▪ Amphotericin B (most widely used) ▪ Azoles ▪ Nystatin ● When you stop ergosterol synthesis you stop the infection from continuing. Fungi 3 types ● Moulds ● Yeasts o Are not gram positive, no outer membrane or peptidoglycan ● Thermally dimorphic (primary pathogens o o Mould at 20 degrees Yeast at 37 degrees 1. Body of fungi called a thallus (masses of hyphae) 1. E.g. the white powdery layer on moldy fruit Molds 1. 0. Yeasts 2. 2. Sometimes grow as different colors 1. 2. 3. 4. Oval or spherical Reproduce by budding If buds fail to detach form pseudophyphae not a real hyphae Candida albicans may attach to epithelial cells as yeast but invades deeper tissues by means of pseudohyphae 1. 2. 3. 0. Yeast can also grow in the mouth and when it does its called thrush 1. Saccharomyces - yeast, ethanol for wine, carbon dioxide for leaving bread dough - nonpathogenic 1. Yeasts are capable of facultative anaerobic growth, most moulds are aerobic only (important for invasiveness) 1. Cryptococcus neofromans 1. 1. 2. 0. Thermally dimorphic fungi 1. 2. Found in pigeon poop Produce a polysaccharide capsule Two forms of growth either a mold or a yeast (mold → more infectious) 1. Once turned into a yeast cannot pass from person to person Dimorphism → temperature dependent. Pathogenic species grow as moulds at lower temps and yeast at body temps 1. 1. 2. Dimorphic fungi are not infectious from one person to another - the mould form is spread by spores in the air Primary pathogens so everyone gets infected but not everyone gets sick 1. Can prove they have been infected if you look for antibodies in the blood 1. Sporothrix (subcutaneous infection) ● Dimorphic also called “Rose Gardener’s Disease” ● Fungal spores introduced into wound after trauma o Does not get into the bloodstream or disseminates ● Lymphangitis common (usually arm or leg) - causes more ulcers as disease progressed o Lymphangitis → inflammation of the walls of the lymphatic vessels Fungal reproduction ● Formation of spores (not real spores like bacteria) o Conidia → important for microscopic identification and for reproduction ● Very different from bacterial endospores ● Fungal nutrition ● Less nutritionally demanding than bacteria ● Grow better than bacteria in extreme conditions ● Most are aerobic and grow best at 25-30 degrees Fungal diseases ● All fungal diseases are called mycoses (plural) and mycosis (singular) ● Types of mycoses o o o Superficial → less severe Cutaneous Systemic → most severe 1. Superficial mycoses 1. Fungal infection along surface of hair shafts and outer layer of skin 2. Mild infections 1. Black and white piedra (hairs of scalp) 2. Malassezia furfur → common cause = yeast) 1. Cutaneous mycoses 1. Fungal infection deeper layers of the epidermis 1. E.g. athletes foot or ringworm 2. Caused by dermatophytes (degrade and use keratin as carbon source - food) 3. 3 different moulds, same mechanism of pathogenicity 4. Clinical diseases are called tineas: 1. Tinea capitis → head 2. Tinea pedis → feet 3. Tinea cruris → groin 4. Tinea corporis → torso 5. Tinea barbae → beard 5. 1. Systemic Mycoses 1. Infections in deep tissues (organs) 1. Caused by primary pathogens such as the dimorphic fungi 2. Or opportunistic pathogens such as Candida albicans. And Aspergillus species 1. Opportunistic fungal pathogens 1. Fungi are common cause of infections in immunocompromised individuals 1. Aspergillosis: caused by Aspergillus species 1. Infects people with debilitating lung diseases and immunocompromised individuals 2. 3. 1. Candidiasis 1. Candida albicans 1. Causes vulvo-vaginal candidiasis and thrush (occurs in newborns, immunocompromised and in people who have been treated with broad spectrum antibiotics) 1. C.albicans is normal flora in the gut and mouth 2. Part 2 infection control Objectives ● Define common terms used to describe disinfection sterilization, pasteurization, sanitization and antisepsis ● Identify uses for positive and negative pressures in isolation rooms ● Describe the process of sterilization and control of the process using the endospore test ● Explain why alcohol based sanitizers are not appropriate for naked viruses and endospore containing Terms to know: chapter 6 table 1 1. 2. Antiseptic → disinfectant used on the skin Aseptic technique → methods to exclude microorganisms 1. Septic means infected 3. Bactericidal → kills bacteria 4. Bacteriostatic → inhibits growth of bacteria, does not kill 5. Disinfectant → chemical used to destroy many micro-organisms and viruses 6. Fungicide → kills fungi 7. Pasteurization → Brief heat treatment used to reduce the numbers of organisms and to kill pathogenic organisms 8. Sanitization → reduction of the number of organisms to a level that meets public health standards ( not a concept in healthcare) 9. Sterilization → killing all forms of microorganisms, including “inactivating” viruses and spores (really important in healthcare, once you reactivate a spore or virus → cannot reactivate and this is key) (Need to use sterile equipment in surgery) 10. Viricide → inactivate viruses ● Ventilation Control the environment o Ultra-clean air ▪ o High-efficiency particulate air filters (HEPA) Building standards for correct air flows o Isolation rooms with “air locks” ▪ Positive pressure → air from corridor cannot get into room ▪ If patent is very susceptible to infection (patient with a stem cell transplant) ▪ ▪ Air travels from least pressure to most pressure Negative pressure → air from room cannot get into the corridor ▪ Air around patient cannot get into corridor ▪ A patient with TB or measles or if they are very infected ▪ Air from hallway into the room not from room into the hallway Physical means of controlling microbial growth ● Heat o Hot air (160-180 degree celsius) for 1-2 hours ▪ Autoclaving (moist heat) → 121 degrees celsius, 15psi ▪ ▪ Will kill C,botulinum spores (industry standard) Test for effective sterilization: “spore test” using Bacillus stearothermophilus spores (test for inactivation) ▪ o Irradiation (gamma or UV) o Filtration o Chemicals o Most important consideration that determine the efficiency of sterlization is whether or not the object to be sterilized is free of organic matter (blood, fecal material, tissues) ▪ Best to rinse / wash before sterilizing ● Filtration ● Cold ● High pressure ● Desiccation (drying) ● Osmotic pressure ● Radiation Spore test ● Based on testing the ability of the autoclaving procedure to totally inactivate bacterial spores o Ampule containing nutrient media and spores of a non-pathogenic bacteria are autoclaved with object to be sterilized.The ampule is then incubated to test for growth o Growth (lack of sterilization) is indicated by a change in the pH of the ampule growth media seen as a color change as the spore has germinated and the growing or vegetative bacteria is using nutrients in the medium and producing acid or alkali compounds. o o Positive test → not sterile (pH of the medium changed because bacteria are growing) Negative test → sterile ● Chemical means of controlling growth (only few of these are sterile) 1. Hand Soap → surface active agents 2. Phenols → good disinfectants, remain active in presence or organic material 1. Lysol 3. Bisphenol → antiseptic 1. Phisohex 4. Biguanides → antiseptic 1. Chlorhexidine 5. Quaternary ammonium compounds 1. Cepacol 6. Alcohols 1. Isopropyl/ ethanol 7. Heavy metals → silver and mercury ● Red → STERILIZES 0. 0. 0. 0. Most used and useful (disinfectants” in everyday health care situations Halogens → chlorine, iodine (can sterilize if used well) Aldehydes → sterilize 1. Glutaraldehyde or Cidex 1. Often used in hospitals Ethylene oxide → gas that sterilizes Preroxygens → strong oxidizer, sterilizes. ● Halogens → iodine and chlorine o Iodine → available as tincture (sol’n in alcohol) or as an iodaphor (comb, of iodine and organic molecule which releases iodine slowly) e.g. Betadine ▪ o Chlorine → strong oxidizing agent e.g. Chlorox ▪ o ● Alcohols o o o o o o Betadine → used for pre-surgical preparations New for backpackers: chlorine dioxide purported to have good effect for decontamination of water and leaves no residual chlorine taste to water Note → halogens can sterilize if appropriate amount of active chemical is used and prolonged time of exposure. Kill bacteria and fungi but not endospores and non-enveloped viruses Mechanism is protein denaturation and disruption of the lipid membranes Used for skin “degerming” Not good for treating wounds as they cause coagulation of proteins, creating an environment where the bacteria can grow inside the scab Optimal conc. Ethanol → 70% (denaturation needs water to work) Isopropanol → rubbing alcohol → better than ethanol (does not evaporate as fast) Relative resistance of microorganisms to chemical agents ● Prions needs to be autoclaved for 2 hours at 160 degrees after soaked in normal sodium hydroxide for 4 hours these are very hardy Summary for part 2 ● Sterilization: inactivation of spores ● Antisepsis : for skin ● Disinfection: for surfaces ● Physical vs chemical disinfection ● Not all methods are appropriate for mycobacteria and spore inactivation! MMI 133: Lecture 8 Antimicrobials ● Learning Objectives ● Define: bactericidal and bacteriostatic, spectrum of activity, broad-spectrum drugs, drug of choice, MIC and SIR. ● Identify mechanisms of antibiotic resistance ● List five modes of activity of antimicrobials and learn the examples given ● List some problems with chemotherapy for bacteria, viruses, fungi and parasites ● Explain why monitoring the level of some antimicrobial drugs in a patients serum is necessary ● List the common superbugs and identify the reason they are classed as superbugs Early “ antimicrobial substances” 1. 2. Damaged the host as much or more than the pathogen 1. E.g. early syphilis treatment 1. Mercury → 15th century 1. “ a night with Venus a lifetime with mercury” 2. Arsenic → 1910 1. “ magic bullet” 3. Fever → 1917 1. Induced malaria → neurosyphilis 1. When we get a fever → temp raises and bacteria might stop reproducing if they do not like the temp. 4. Penicillin → 1929 (seen as the magic bullet with selective activity on prokaryotes) 1. Worked only on prokaryotes not eukaryotic cells 2. Alexander flemming 1st synthetic antimicrobial substance to act selectively on bacteria 1. SULFA (1932 - prontosil) Antibiotic era 1. : PENICILLIN → Hailed as “wonder drug” 1. Alexander Fleming 1. Penicillin → 1929 2. Failed at isolating and purifying the drug 2. Howard Florey 3. Ernst Chain 1. 1. Both isolated penicillin from solution of mold that was growing Why don’t antibiotics kill human cells 1. The middle → structures that are similar in both, so any antibiotic that targets any of the things in the middle can harm eukaryotic cells. What weapons do we have? 1. 2. 2. Antiparasitic agents 3. Antifungal agents 4. Antiviral agents 5. Antibiotic 6. Some immunoactive substances e.g. interferon can make people feel very sick 7. Maintenance of immune integrity, hygiene Antiparasitic agents: antiprotozoan and anti-helminthic drugs 8. *Quinine → derivatives for Malaria 1. Problem → increasing resistance of malaria to the drugs Anti-fungal agents 9. Problem with toxicity → fungi and eukaryotic organisms 10. Most succesful aganets affect the plasma membrane of the fungi 1. Contains ergosterol instead of cholesterol 11. All drugs have some toxicity (kidney) 12. Most common durg → amphotericin B (inhibits ergosterol synthesis in the cytoplasmic membrane of the fungal cell 1. Has a bad effect on kidney Anti-viral agents 1. 2. 3. 4. 5. Bacteriostatic vs bactericidal 1. Bactericidal DNA/RNA synthesis inhibitors Entry inhibitors (blocking the attachment of the virus to the host cell) Uncoating inhibitor → influenza is an example (gets down to cytoplasmic cell and is in a vacole so it clogs up theM2 proton channel Nucleoside analogue 1. Azidothymidine (AZT) or (Zidovudine) Treats HIV 1. Inhibits the reverse transcriptase (synthesis of DNA from RNA) 2. There was a mix up between thymidine and AZT, if mixed up the AZT it would not work Protease inhibitors Bacteriostatic 1. Stop the replication of bacteria 2. Do not kill the bacteria already present 1. Erythromycin → macrolides 1. The immune system has to use any kind of drug that we use to treat. As long as immune system is working fine to help 1. 2. Kills bacteria Stop bacterial metabolism 1. Penicillin (beta-lactam) 1. Prevents peptidoglycan production 2. When someone has a bloodstream infection we want to kill it immediately. 1. Bugs and drugs app → useful Antibiotics and how they work Major classes of antimicrobials 1. Know these and common examples 1. Inhibition of cell wall synthesis 2. Inhibition of protein synthesis 3. Injury to plasma membrane 1. Topical antibiotics 4. Inhibit nucleic acid synthesis 5. Inhibit synthesis of essential metabolites Specific antibiotics 1. 2. Inhibit Gram - OR Gram + organisms OR certain bacterial species Broad-spectrum antibiotics → inhibit both Gram - and Gram + bacteria 1. Can start broad if don’t know and then go more specific once you figure out what the bacteria is Gram - have 2 membranes, Gram + only have 1 3. The most selective antimicrobial activity would be exhibited by a drug that → just prevents cell wall synthesis Knowing the cause 1. 2. If you know the cause of an infection, you should use as narrow a spectrum activity you should use a narrow spectrum drug More broad spectrum → amoxicillin Who are more susceptible to bacterial infection? 1. 2. 3. 4. 5. Diabetics 1. Hard to heal skin infections Children 1. Developing immunity Elderly 1. Immune Essence Burn wound: skin and soft tissues 1. “Sterile” tissues → exposed Immunocompromised 1. Deficient immune system, neutropenia (missing most important white blood cell) and chemotherapy ● Lack of hygiene can cause infection in anyone Are all antibiotics harmless 1. 2. 3. 4. Allergies → penicillin Pregnant women → tetracycline Children → different pharmacokinetic and pharmacodynamic issues People with liver or kidney damage → Liver: fluoroquinolones, macrolides, Kidneys: aminoglycosides Antimicrobial side effects 1. * do not need to memorize for knowledge 1. Kidney - aminoglycosides 2. • Liver - fluoroquinolones 3. • Rash, allergy - penicillins 4. • GI upsets - penicillins 5. • Nerve damage - fluoroquinolones 6. • Ototoxicity - aminoglycosides 7. • Teeth and bone formation in children - tetracyclines 8. • Cartilage formation in children – fluoroquinolones No antibiotic will be effective is used too late in the infection 1. 2. 3. 4. Too many bacteria Too much tissue damage Formation of walled-off abscesses that can’t be penetrated by antibiotics Poor absorption of drug Why are IV antibiotics used instead of oral 1. 2. 3. 4. They go directly into the blood stream GI problems Only drug possible → vancomycin (only available in IV solutions) Rapid bioavailability: 1. Serious infections: meningitis, septicaemia, endocarditis Overwhelming infection of Clostridium perfringens 1. Pseudomonas aureginosa (Gram -) 1. 2. Found in soil/ water Opportunistic pathogens → can cause really damaging effects 1. Can even live in liquid soaps, water 2. Needs very few nutrients 3. Very common in burn patients and patients with cystic fibrosis 1. Most people with cystic fibrosis → mucus makes it easier for bacteria to grow 4. Has endotoxin also produces exotoxins 1. They can cause progressive tissue damage 2. Resistant to a lot of drugs (a lo of antibiotics come from soil organisms) Polymicrobial infections 1. 2. Caused by many bacteria working together at the same time Do not know the bacterial type 1. Enterobacter 2. Staphylococcus 3. Streptococcus Penicillin (the wonder drug) 1. is discovered and used successfully for a time (very good for staphylococci) But- then the bacteria rebelled… 1. Bacterial mutation resulting in: 1. Production of the enzyme beta-lactamase or penicillinase 1. Inactiviation of penicillin by breaking ‘the beta-lactam ring” 2. There is a certain structure in penicillin called the beta-lactam ring, essential for it to stay together 3. 4. How do bacteria become resistant to antibiotics R-factors 1. 1. Synthesis of semisynthetic penicillins with a structure where active portion of the antibiotic was protected from the enzymes 4 ways 1. 2. 3. 4. Prevent penetration Destruction of drug Target site alteration Ejection of drug from bacterium (drug efflux) - Plasmids that confer genes for resistance to antimicrobials 1. Genes coded on small circular independently replicating pieces of DNA called plasmids located in the cytoplasm of bacteria 2. Can be transferred from one bacteria to another by conjugation (sex pilus) 3. R-factors can be transferred between different bacterial species 1. E.coli to Shigella Why do bugs become resistant? 1. 2. 3. Overuse Inappropriate treatment Incomplete treatment regimens What are the heck are superbugs? 1. Bacteria that cannot be controlled by antibiotics MRSA = methicillin resistant S. aureus Note MSSA = methicillin susceptible S. aureus o o o Changed the penicillin binding site on the bacterium to produce a variant that was resistant to the methicillin Penicillin binding protein 2 (PBP 2) mutates to PBP2a (encoded by MecA ) = low affinity for binding penicillin Penicillin then will not be effective for inhibiting the cross-linking of peptidoglycan….. Treatment remaining is: vancomycin. ● The first isolate of VRSA also resistant to methicillin (and oxacillin) has appeared. ● VRE (Vancomycin Resistant Enterococci) o Genetic mutations - several in genes Van A, Van B, Van C ▪ Resistance developed after use of “avoparcin” glycopeptide in animal feed ESBL (Extended Spectrum BetaLactamase) 1. 2. 3. 4. 5. Enterobacteriaceae like E. coli: genetic mutations, many resistance genes Increasing problem in ICU, extended care Resistance to penicillins and cephalosporins ESBL can inactivate even the new really good beta-lactams like meropenem NDM-1 (New Delhi metallobetalactamase is one of the latest enzyme variants) XDR - TB 1. 2. 3. First seen in Tugela Ferry, a rural town in South Africa in 2006 53 patients, 52 died, median age 35 yr Resistant to both first and second line anti-TB drugs 1. New hope → Pretomanid + Bedaquiline + Linezolid for 6 months (90-95% cure rate) MIC = Minimum Inhibitory Concentration 1. Minimum amount of antimicrobial that will inhibit the growth (( not kill) the microorganism 1. Appropriate dosing levels → very important Every important bacterial isolate must be tested Need to know for appropriate dosing levels and intervals Drug of choice 1. Drug that is deemed to be most effective with the least toxicity for a specific infection 1. E.g. penicllin is the “drug of choice” for streptococcal infections in throat “pharyngitis” Disk diffusion test for antimicrobial susceptibility 1. Routine test used in laboratory, primarily for rapidly growing bacteria as the antibiotics on the discs will deteriorate with time. Three categories of sensitivity are defined: 1. S = sensitive or susceptible 2. I = intermediate 3. R = resistant Kirby Bauer (or disk diffusion) susceptibility test 1. 2. Interpretation: measure the zones of inhibition and compare to standard charts to get S,I,R This is a one shot concentration of the antibiotic in each disc (based on prescribing standards). You use this for infections like urinary tract infections, skin infections, but not for infections in sterile sites (like blood). E-test (Ellipse test) for MIC determination 1. An easy way to determine MIC for a drug on a specific bacterial stain. 2. 3. 2. Monitoring of serum antimicrobial levels 1. Needed for some drugs and some types of patients 1. Attain effective levels (Over MIC) 2. Prevent toxic side effects 1. Chloramphenicol → issues with bone marrow 2. Aminoglycosides → ototoxic and nephrotoxic. May cause permanent damage (deafness and kidney dysfunction) 3. Ascertain dosing intervals (every 8hrs, 12hrs, 24 hours etc) 1. Need to keep track of how long the antibiotics lasts in the blood Summary 1. 2. 3. 4. 5. Neonates, elderly and immunocompromised are the most susceptible to deadly infections . • Bacteria have multiple strategies for drug resistance. • Dangerous superbugs: MRSA, VRE, ESBL and XDR-TB. • Superbugs are a huge threat for the whole health system . • MIC and SIR: Personalize and control the dose.