Bacteriology and Parasitology Notes PDF

Introduction to bacteriology Current view of the tree of life: - 3 domains: bacteria, archaea, eukaryotes - Bacteria and archaea are prokaryotes - 30-40 years ago it was believed that bacteria and archaea were in the same domain Prokaryotes - The smallest, simplest, and most ab...

Introduction to bacteriology Current view of the tree of life: - 3 domains: bacteria, archaea, eukaryotes - Bacteria and archaea are prokaryotes - 30-40 years ago it was believed that bacteria and archaea were in the same domain Prokaryotes - The smallest, simplest, and most abundant cells on earth - Prokaryotes include bacteria and archaea - Prokaryotes lack a nucleus and complex organelles - No mitochondria, ER, etc Bacteria can grow fast - Bacteria reproduce asexually by binary fission (do not exchange DNA) - 4 phases of growth: - Lag phase: adapting to the medium - Logarithmic growth: exponential growth of cells - Stationary: no growth (period of stability) - Death: cells are dying due to competition (not enough resources for all of them to survive - Generation time (doubling time) is highly variable between bacteria species - The amount of time for 1 generation - Some bacteria are very fast growers, and some are very slow - Some species such as E.coli grow fast (double in ~10 minutes) - Some species such as mycobacterium tuberculosis grow slow (double in ~24 hours) - The growing time does not affect the survival of bacteria Bacterial classification by shape - Bacteria are usually named based on their shape - Coccus (round shape) - Bacillus (rod shape) - Spirillum (spiral shape) Bacterial classification by O2 utilization - Obligate aerobe: require oxygen for growth - Obligate anaerobe: oxygen is toxic for growth - Facultative anaerobe: can use oxygen is present, but can also grow without oxygen - Aerotolerant anaerobe: does not use oxygen, but is also not toxic to it - Microaerophile: grows best with low levels of oxygen (small amounts) Taxonomic ranks (example: e.coli) Domain: bacteria Kingdom: Eubacteria Phylum: Proteobacteria Class: gammaproteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genus: Escherichia Species: coli Strain: O157:H7 - Name is genus (written in uppercase) and species (written in lowercase) in italics E.coli genomes are much more diverse than humans despite being the same species - 2 e.coli strains can be only 60% similar - All humans are 99.5% identical Basic bacterial cellular structure - Cytoplasm - Nucleoid (contains the chromosomes) - Ribosomes - Plasmid - Cytoplasmic membrane - Cell wall - Cell envelope (outside cytoplasmic membrane) Gram staining - To distinguish between 2 different types of bacteria - Gram positive = purple - Gram negative = pink 1. All cells are placed on a glass slide and smeared with crystal violet for 1 minute - All cells appear purple 2. All cells are counterstained with iodine solution - All cells remain purple 3. 70% ethanol solution is used to decolorize the cells - Gram positive cells remain purple, gram negative cells become colourless 4. Cells are counterstained with safranin - Gram positive cells are purple, gram negative cells become pink - Some bacteria are neither gram positive or negative and require different staining procedures to view under a microscope - Acid fast staining: mycobacteria - No cell wall: mycoplasma Gram positive vs. gram negative bacteria Gram positive - Contain a thick cell wall made up of peptidoglycan and teichoic acids that surrounds the cytoplasmic membrane - Thick cell wall retains the gram stain color when decolorized Gram negative - Have a thinner layer of peptidoglycan in the inner cell wall - Have 2 cytoplasmic membranes - Outer membrane contains lipoproteins and lipopolysaccharide - The thinner cell wall does not retain crystal violet stain very well, and is washed off by ethanol Organelles in bacteria Bacterial cell walls - Called peptidoglycan - Rigid structure - Prevents osmotic lysis - Glycan backbone: - N-acetylglucosamine (G) - N-acetylmuramic acid (M) - Peptide cross linkage - Peptide bonds alternate between G and M, and are crosslinked by N-acetylmuramic acid Lipopolysaccharide - O-specific polysaccharide (also called O-antigen) (endotoxin) - Antigenic and highly variable - Core is made up of polysaccharide and lipid A - Disaccharide + fatty acid groups - These are recognized by the immune system (PAMPs) - Can lead to septic shock - Toll-like receptor 4 would recognize this - Core is not antigenic and is generally conserved through bacteria Nucleoid - Is not a nucleus - No surrounding membrane - Single, circular chromosome (most but not all bacteria) - Haploid genomes (one set of chromosome) Plasmids - Extrachromosomal genetic elements - Usually not required for bacterial growth - Often encode for ‘fitness’ factors - Example: antibiotic resistance - Useful in cloning experiments  - Can be transferred from bacteria to bacteria (horizontal gene transfer) The human microbiota - Internal organs are usually sterile - Surface tissues have extensive populations of microbes - The collective genome of the human microbiota easily contains more than 100 times as many genes as our own genome - More microbes living in our body than our own cells - The types of bacteria depend on the tissue/location in the body Host-microbe relationships - Commensalism: one benefits without hurting or helping the other - Mutualism: both species benefit (the host and the microbe) - Parasitism: one benefits (usually the microbe) at the expense of the other (host) Bacterial pathogens - Colonization: bacteria multiply inside the host - Invasion/toxicity: cause problems in host by producing toxins - Immune evasion: hide from the host immune system - Transmission: find another host - Pathogens produce virulence factors: molecules produced by the pathogen that contribute to disease Virulence factors - Surface - LPS (endotoxin) - Flagella - Pili and adhesins: used to stick to surface - Capsules: protective lining outside cell wall or LPS - Secretion systems - Secreted - Exotoxins - Example: tetanus can produce an exotoxin that can result in paralysis Flagella - Structures that allow some bacteria to be motile (chemotaxis) - Counterclockwise rotation produces run (forward movement) - Clockwise rotation produces tumble (allows bacteria to change direction) Pili - Primarily involved in attachment to surfaces, host tissue, and other bacteria - Surfaces include plaque that forms on teeth  Capsules - Usually made of (exo)polysaccharides which are quite thick - Attachment to host tissues - Protection from host immune system - Cytokines cannot get through this easily - Can sometimes be used in vaccines - Formation of biofilms Biofilms - A complex structure that can produce toxins and release cells - Stages: - Attachment: a single cell attaches to a surface - Microcolony development: cell replicates to form a colony - Biofilm development: cells form a capsule around the entire colony by producing exopolysaccharide (EPS) - Maturation: the biofilm is fully created, made up of multiple complex structures - Dissolution/dispersal: releases cells and toxins which can go on to potentially make another biofilm - In some cases, biofilms can be seen without a microscope, such as on surfaces like kidney implants Endospores - Highly differentiated cells formed within the parent cell - Highly resistant to heat, harsh chemicals, and radiation - A ‘dormant’ stage of the life cycle - Can survive for decades in this form - Most common in: - Soil - Bacillus and clostridium genera - Generally only formed by gram positive bacteria Exotoxins - Secreted from bacteria - Includes: - Hemolysins: breaks down RBCs - Toxins that function inside host cells (intracellular) - Extracellular enzymes that contribute to proteolysis - Superantigens: activate T-cells in the host - Some exotoxins (inactivated form) can be used as vaccines - Example: tetanus vaccine Bacteria as intracellular pathogens - Are taken up and survive within phagocytic cells (such as macrophages) - Some ‘force’ their own uptake into epithelial cells - Allows bacteria to hide from different components of the immune system - Example: mycobacterium tuberculosis The black death - Gram negative, rod shaped bacterium - 3 species are pathogenic for humans: - Y. enterocolitica – causes "yersiniosis" – a rare cause of diarrhea and abdominal pain - Y. pseudotuberculosis – primarily an animal pathogen that can cause tuberculosis-like symptoms in animals, enteritis in humans - Does not cause tuberculosis in humans - Y. pestis – cause of Plague Yersinia pestis - Discovered by Alexandre Yersin and Kitasato Shibasaburo in the late 1800s - pestis -> pestilence (contagious or infectious epidemic disease) - an extraordinarily virulent pathogen compared to other bacteria - May cause death in 2-4days by sepsis and/or overwhelming pneumonia with respiratory failure: most aggressive form of disease - Can spread via aerosols - NOT an efficient colonizer of humans - Does not colonize skin, but rather immune cells Plague - Incubation of 3-7 days - Death may occur in 2-4 days - Patients experience sudden onset of fever, chills, headaches, muscle pain, weakness - Painful swellings (buboes) of the lymph nodes in the armpits, legs, neck, or groin - This is where the name “bubonic plague comes from - High fever, delirium and mental deterioration, large blacking pustules that burst, vomiting of blood (during the pulmonary phase), bleeding in the lungs Plague pandemics - The plague of Justinian (the first pandemic) - Named after the Eastern Roman emperor Justinian - Started in the 6th century (541-542 AD) - Caused by Yersinia pestis - Spread to the mediterranean, Italy and throughout Europe - 50% of the population is estimated to have died - Continued in cycles for another 200 years until about 750 AD then disappears for 800 years - Overall, it is estimated to have killed 100 million people - The black death (the second pandemic) - A medieval pandemic caused by Yersinia pestis - Originated in Asia and reached europe in the late 1340s - Reduced the global population from ~450 million to ~350 million - Killed ~25 million Europeans (⅓ of the total population) - Plague doctors: pretending to treat people (not real doctors) - Mask was stuffed with flowers and herbs which were thought to repel the disease  - People had no idea what was happening (no knowledge of bacteriology) - No (real) treatment; bloodletting was used - Many people believed that this was due to God’s anger or satan’s influence - Persecution of strangers, minorities, and witches - European social order, family structure, agriculture, military and feudal system was destroyed - The feudal system - Political and social structure prevalent in europe - Kings -> Nobles -> knights -> peasants - People at the bottom of the structure had little to no opportunity for advancement - A few people at the top had everything; most had very little - Plague; created vacant towns and farms - Demand for physicians, clergy, gravediggers - Provided new opportunities for the peasants - We know Y. pestis caused the pandemic because graves of plague victims were dug up and samples of Y. pestis were collected - Next generation sequencing was used to partially sequence the genome of Y. pestis - A different strain of Y. pestis than the one that exists today caused the black death - Mid-19th century (the third pandemic) - Started in china in the 1850s and spread to all continents - considered to have ended in 1959 - 12 million deaths in china and india alone - Reached san francisco in 1900 infected rats exchanged fleas with local wildlife - Y. pestis is now established in southwestern U.S. Pathogenesis of Yersinia pestis - Organisms live in rodents and are transmitted by fleas - A zoonotic pathogen - Y. pestis causes blocking in the flea - Creates a biofilm in the gut of the flea, which causes it to starve because it cannot digest the blood - This causes the flea to throw up the stomach contents, which includes the bacteria - Very low infective dose (~10 cells) - Y. pestis initially survives and grows in innate immune cells (macrophages, etc) - Replicates in lymphoid organs (spleen, bone marrow, lymph nodes, liver) - Lymph nodes: swelling (buboes) - Y. pestis kills phagocytes and continues to grow extracellularly - At the terminal stage of the disease, the blood contains high concentration of bacterial cells - Essential for transmission as fleas take a blood meal Virulence factors of Yersinia pestis - Can overcome immune defense mechanisms by growing in vivo (inside cells) - Can evade the mammalian innate immune response - Allowing for infection before it even gets noticed by the immune system - Major virulence factors: - Type III secretion: typical for gram negative intracellular pathogens - Phospholipase: for survival inside the flea - Plasminogen activator: activates host protein plasminogen which is a clot buster - Critical for infection spreading to other parts of the body - Yersiniabactin: iron binding siderophore - LPS structure is mutated in Y. pestis: makes it hard for the immune system to recognize it - Generally, LPS can be easily detected by the immune system - Classified as a level 3 pathogen - Level 3 pathogen in level 2 lab: strain that is used must be attenuated to reduce virulence factors - Level 3 labs do exist, but they are much more costly to build and maintain - If strain is not attenuated, it can cause a major outbreak if protocol is not followed Type III secretion systems - Found in many gram negative pathogens (usually intracellular pathogens) - Secrete virulence factors (called effectors) directly into host cells across the host cell membrane - Effectors function to poison the host cell by targeting host cell signaling pathways - Bursts out of cell once it multiplies - A microscopic syringe that injects toxins into the cell Evolution of Yersinia pestis - Y. pestis evolved from Y. pseudotuberculosis - Acquired new virulence plasmids (for encoding phospholipase D and plasminogen activator) - Also acquired chromosomal genes for biofilm formation and insect toxins - All pathogenic Yersinia contain pYV which encodes the type III secretion system - Y. pseudotuberculosis is primarily an intestinal pathogen of animals and is found widely in the environment - Y. pestis can infect the flea and is hypervirulent in humans, but does not survive well in the animal intestine - The LPS of Y. pestis is weakly recognized by the innate immune system due to a mutation in lipid A modifying enzyme Plague - 3 major forms of the disease: - Bubonic plague - Most common form, transmitted by flea bites - Painfully swollen lymph nodes (buboes) in groin, armpits and neck  - Can develop into both septicemic and pneumonic plague - 40-60% mortality if untreated - Septicemic plague - Presence of Y. pestis in systemic circulation (in the blood) - An overwhelming and progressive bacteremia - Fleas can now pick up Y. pestis to transmit it to a new host - Patients experience gangrene and disseminated intravascular coagulation - 50-90% mortality if untreated - Pneumonic plague - Most dangerous - Transmission via aerosols directly into the lung, or spread into the lungs from septicemic plague - Short incubation - Disease can pass directly from person to person through coughing (coughing up blood) - 95-100% mortality if untreated, but treatment must be within first 24 hours of symptoms Transmission - 4 routes for human disease - Flea bite (most common) - Inhalation from humans or animals (pnemonic) - Handling infected animals: skin contact, scratch, bite - Ingesting infected meat - Historically, rat-borne urban epidemics - Now mostly wildlife associated plague with sporadic outbreaks Diagnosis, treatment and prevention - Rapid diagnosis and treatment is essential due to aggressive nature - Culture and identification from bubo aspirate, sputum, blood (post-mortem) may take 4 days - In endemic regions, there are stains and rapid antigen tests - Isolation of pneumonic plague patients - Insecticides are used to kill fleas, human cases treated with appropriate antibiotics including prophylaxis to exposed individuals - Used to control spread Plague as a bioterrorism agent - The center for disease control and prevention identify plague as a category A organism - Can be easily disseminated or transmitted from person to person - Result in high mortality rates and have the potential for major public health impact - Might cause public panic and social disruption - Y. pestis is easy to grow - Known incidents:  - The mongol armies threw plague-ridden bodies over the city walls in Caffa, Ukraine - In WWII, the japanese imperial army released infected fleas in China Plague outbreak in Madagascar - Large outbreak starting in August 2017 - 2400 probable/confirmed cases, most were pneumonic - ~200 deaths - Not as many people died because this is an endemic region and preventative measures were already in place Antibiotics and antibiotic resistance Antimicrobial agents - Disinfectants: antimicrobial agents that are applied to inanimate objects (floors, tables, walls, etc) - Antiseptics: antimicrobial agents that are sufficiently nontoxic to be applied to living tissues (hand sanitizers) - Antibiotics: antimicrobial agents that are produced by bacteria and fungi that are exploited by humans (delivered topically and internally) - bacteria/fungi produce antibiotics to compete for resources - Does not target your cells: only works on bacteria Antibiotics - Antibiotics represent our most effective therapeutic against bacterial infections - The availability of antibiotic enables cancer chemotherapy, organ transplantation, all invasive surgeries, treatment of premature infants - 2 major problems: - Diminished interest from pharmaceutical companies to develop new antibiotics - Bacterial resistance to antibiotics always happens Misuse of antibiotics - Empiric use (blinded use); when you use antibiotics on every infection - Increased use of broad-spectrum agents; drugs that can be used to treat multiple diseases - Pediatric use for viral infections; have no effect on viruses - Patients who do not complete course (chronic disease, e.g. TB) - On drug for 6 months, symptoms get better, but return after some time (as TB develops resistance to it) - Antibiotics in animal feeds; to treat infections in animals and release these into the environment - Resistance genes can travel globally very quickly Measuring Antibiotic activity  - Minimum inhibitory concentration (MIC) - Series of liquid culture tubes with varying concentration of agent - Check for visible growth - MIC = lowest concentration of agent that completely inhibits growth - Antibiotic strips - faster , multiple antibiotics - Petri dish with antibiotic strips is left to incubate - Gradient concentration across strip - Most concentration at the top, lowest at the bottom - MIC = the lowest concentration that presents a zone of clearance How do antibiotics work? - Antibiotics target essentia;l bacterial componenets: - Cell wall synthesis - Protein synthesis: ribosomes (different from eukaryotic ribosomes) - DNA/RNA synthesis: disrupts DNA/RNA of bacteria - Folate synthesis: stops the production of folic acid - Cell membrane alteration: breaks cell membrane - Targets are not present (or different) in eukaryotic cells Beta lactam antibiotics - Penicillin - Contains a beta lactam ring (square ring with a nitrogen and a ketone) - Inhibits cell wall synthesis in bacteria - Beta lactams bind to the bacterial “penicillin binding proteins (PBPs)” - PBPs are transpeptidases; used to cross link proteins in the cell wall - Drug prevents crosslinking - No peptide cross-links = weak cell wall = death - Some bacteria can produce a beta lactamase - Disrupts drug and allows bacteria to survive - Methicillin - Contains a beta lactam ring - Chemically modified penicillin - Cannot be cleaved by beta lactamases - Some bacteria can produce a different penicillin binding protein (ex. PBP2a) which is encoded by ‘mec’ (mec gene) - PBP2a does not bind methicillin or any other beta lactams Vancomycin - A glycopeptide antibiotic - Inhibits cell wall synthesis in gram positives - Often a drug of “last resort” (ex. MRSA) - Used to kill bacteria that are resistant to all other available antibiotics  - Vancomycin binds to the peptide linkage at terminal D-Ala-D-Ala residues and inhibits transpeptidation - Resistance genes can change these to D-Ala-D-Lac (lactase), preventing vancomycin from binding - Mutates the peptide that vancomycin binds to - Resistance is encoded by the ‘van’ genes Summary: - Beta lactams inhibit transpeptidases - Beta lactamase cleaves penicillin - Methicillin bypasses beta lactamase and inhibits transpeptidases - PBP2a is another transpeptidase that cannot be inhibited by beta lactams - Vancomycin binds to the peptides themselves, which prevents cross linking - Bacteria can mutate the peptide to prevent vancomycin from binding Selection for antibiotic resistance - The use of antibiotics actively selects for antibiotic resistant bacteria - The bacteria with the resistance genes will survive, and pass on their genes to the clones Strategies for antibiotic resistance in bacteria - Prevention of antibiotic entry - Gram negative outer membrane and mycobacteria cell envelope - Innate resistance to antibiotics; antibiotics cannot pass through the envelope easily - Antibiotic modification - Beta lactamase (enzyme production) - Efflux of antibiotic - Actively pump out the antibiotic - Pumps can identify and pump out the antibiotic - Alteration of antibiotic target - PBPs, ribosome modifications - Mutated target site to prevent binding of antibiotic - Bypassing the antibiotic action - Use environmental folic acid Antibiotic resistance genes - Many mechanisms of antibiotic resistance are genetically encoded (mec, Beta lactamase, efflux pumps) - These are all encoded in the chromosome - Can produce high levels of antibiotic resistance - Resistance genes are often encoded on mobile genetic elements (plasmids) which allow them to pass the genes along through horizontal gene transfer; can result in the creation of “superbugs”: bacteria that have acquired multiple resistance genes Horizontal gene transfer - Rather than alter gene function through mutations, new genes are acquired from another source 3 main ways this occurs: 1. Bacterial transformation - Donor cell bursts and releases chromosomal DNA, which could contain antibiotic resistance, and is picked up by a recipient cell 2. Bacterial transduction - A phage takes up the gene by accident and injects it into a new cell 3. Bacterial conjugation - Donor cell forms a cytoplasmic bridge with another cell and transfers a plasmid which contains the antibiotic resistance gene Klebsiella pneumoniae - Gram negative (has innate resistance to antibiotics) - An important cause of nosocomial pneumonia - Produces a capsule and is commonly resistant to multiple antibiotics - First documented source of NDM-1 - New Delhi metallo-beta-lactamase-1 - Also known as carbapenemase - The enzyme destroys carbapenem - Carbapenem antibiotics are Beta lactams that are resistant to beta lactamase - Broad spectrum drug, often used as a last resort - NDM-1 is now widespread in other gram negatives - CRE (carbapenem resistant enterobacteriaceae - E.coli, shigella, other gram negatives Clostridia - Gram positive, rod shaped, endospore formers (very difficult to treat) - Strict anaerobes, vegetative cells killed by O2 - Generally found in soil, and intestinal tracts of animals - These ones are generally non pathogenic - Important human pathogens - Clostridium difficile: pseudomembranous colitis - Clostridium tetani: tetanus - Clostridium botulinum: botulism - Clostridium perfringens: food borne illness and gas gangrene - Can cause life threatening diseases mediated by exotoxins - Bacteria produce exotoxins Clostridium difficile (C. diff) - Can exist as: asymptomatic carrier state in the large intestine - Cause of mild to moderate diarrhea - Cause of life threatening pseudomembranous colitis (life threatening form) - Often found in nursing homes and hospital environments  - A nosocomial pathogen - Endospores can be very difficult to eradicate from the environment - Cultures from floor, bed pans, toilets, hands, and clothing of medical personnel - Mode of transmission is through the spore: fecal-oral route - Cause of pseudomembranous colitis, and called antibiotic associated diarrhea Pseudomembranous colitis - An inflammatory condition of the large intestine - The most important risk factor is having recently received an antimicrobial agent - Diarrhea, abdominal pain, fever, nausea, dehydration - Symptoms may occur 1-2 days after antibiotics or several weeks after antibiotic is discontinued - Endoscopy can show characteristic lesions (white spots on intestine caused by the bacteria) - Lesions can enlarge to cover substantial portions of inflamed mucosa and can be stripped off (the pseudomembrane) - Microbiota: bacteria growing on your tissues - Some are beneficial, these can also be killed by antibiotics Pseudomembranous colitis - Antibiotics are used to cure infections, but they can also kill the normal microbiota - Suppression of normal microbiota + persistence of C. difficile endospores - After the antibiotic is stopped, spores germinate, overgrowth of C. difficile occurs with production of toxins - Spores survive the antibiotic, but the normal microbiota gets killed off, allowing C. difficile to grow with almost no competition - C. difficile is not considered an invasive bacterium, but the exotoxins cause damage and inflammation to the intestinal lining of the large intestine Clostridioides difficile - C. difficile produces A-B toxins called the large clostridial cytotoxins - A-B serves to designate 2 domains - The A domain: active portion of the toxin that carries the enzymatic activity - The B domain: binding portion of the toxin which binds to host cells to be phagocytosed - A domain functions to inactivate key regulatory proteins in host cells - Causes dysregulation of multiple cell processes including cytoskeletal rearrangements, cell death, and inflammation Diagnosis and treatment - History of antibiotic use, symptoms and laboratory tests to confirm C. difficile - Endoscopy and toxin detection assays - Discontinue inciting antibiotic if still being used (because they are killing the microbiota) - Fluids - Antibiotics more specific to C. diff such as oral vancomycin or I.V. metronidazole - Works better on anaerobic bacteria - Avoid antidiarrheal agents: would cause decreased toxin clearance Fecal microbiota transplantation - A donor fecal sample is taken and inserted into the patient to recover their microbiota - If this is not done, the spores from C. diff can persist and cause recurrent infections - The microbiota prevents C. diff from rapidly multiplying (increases competition) - People with fecal transplants often recover from the disease compared to someone only on antibiotics Tuberculosis and leprosy - Aside from covid, TB is the biggest killer worldwide Mycobacteria - M. tuberculosis - causative agent of tuberculosis - Often called TB for tubercle bacilli - Only found in humans - M. leprae - Causative agent of leprosy in humans - M. bovis - Causes tuberculosis in cows, rarely in humans - Humans can be infected by drinking unpasteurized milk leading to extrapulmonary tuberculosis - M. avium - Can cause a tuberculosis-like illness in humans, particularly in patients with AIDS Tuberculosis - Infection by M. tuberculosis can be latent (cannot spread to other people) or active (can be actively spread to others) - Approximately 2 billion people (¼ of the world's population) likely have latent TB - TB is contagious and spread through the air by people who have active TB - Spreads through aerosols - ~10% of people infected with latent TB will develop active TB in their lifetime - ~1.6 million people die from TB each year - TB is endemic in low income countries such as Southern Africa and Southeast Asia Mycobacterium tuberculosis - Intracellular pathogen (lives within macrophages) after being phagocytosed - Slow generation time of >15 hours (compared to other bacteria like E.coli) - M. tuberculosis can be grown in the lab on specialized media but takes 4-6 weeks to get small colonies - Depends on the strain - Takes a long time to grow in the lab - Mycobacteria have an unusual cell envelope with high concentrations of mycolic acid - Makes the envelope ‘waxy’ which results in selective advantages - Mycolic acids are on the outside of the cell wall - Impermeability to stains and dyes - Gram positive Acid fast stain - The unusual cell envelope is associated with resistance to: - Some antibiotics - Osmotic lysis via complement deposition - Lethal oxidative stress promoting survival inside of macrophages - This is how they survive inside - Waxy, lipid rich cell envelope resists common stains due to its highly hydrophobic nature - Gram staining does not work; requires acid fast staining - Acid-fastness is due to the presence of mycolic acid - Acid fast staining is used 1. Cells are stained with carbol-fuchsin dye with slow heating (to melt the wax) 2. Cells are washed with ethanol and HCl 3. All mycobacteria will stain red, rest of the cells will be colourless 4. Counterstained with methylene blue; for cells that are not red 5. Acid-fast organisms appear red whereas non-acid fast organisms appear blue Spread and progression of tuberculosis 1. Transmission is from inhalation of droplets from an infected host, usually by coughing or sneezing - coughing/sneezing can generate 3000 droplet nuclei; droplet nuclei can contain purpuric rash - rash doesn't fade under pressure ("glass test”) - Pushing glass against rash does not make it disappear (normal rashes do) - Serogroup B is the most reported in Canada Vaccines for Neisseria meningitidis - Menactra/menveo - Quadrivalent conjugate capsule vaccine from 4 serotypes of meningococcus (serogroup A, C, Y, W-135) - Serogroup B is the most common for invasive disease in Canada but the capsule is poorly immunogenic (poor immune response) - Bexsero - Contains 4 recombinant protein antigens - Still not as protective as menactra The african meningitis belt - The highest burden of the disease in the world - Can be >1000/100000 - Mostly caused by serogroup A - Sub saharan region where it is endemic - Mostly serogroup A compared to the rest of the world (serogroup B) because vaccines aren’t profitable - Where vaccines are distributed, there is a 95% drop in cases because the vaccine prevents incubation of the bacteria Streptococcus pneumoniae - The ”Pneumococcus" - Gram-positive cocci, grows in chains - Commonly resides asymptomatically in the nasopharynx - Causes pneumonia, ear infections, sinusitis and many other diseases - The leading cause of bacterial meningitis in children > 2 years and adults - Produces a polysaccharide capsule - Many (>90) different serotypes exist - Difficult to make vaccines for all 90 types - A major global pathogen with > 700,000 deaths per year globally Vaccine for streptococcus pneumoniae  - A pneumococcal vaccine has been licensed for use in Canada - Previously a 7 serotype vaccine (PCV7) (used 7 serogroups) - now Prevnar 13 (PCV13) - a conjugate capsule vaccine from the 13 most prevalent serotypes of pneumococcus - use of the vaccine is associated with decreasing rates of invasive pneumococcal disease in Canada and elsewhere - Prevnar 20 is now being developed as well - 23-valent polysaccharide vaccine for high-risk adults, but poorly immunogenic in children (T cell-independent) - Short term immunity Haeomophilus influenzae type b (Hib) - Gram negative, coccobacillus, produces a polysaccharide capsule - primarily causes meningitis in children under 5 - when it occurs, it tends to follow an upper respiratory infection, ear infection or sinusitis - Hib conjugate vaccine available as part of the routine childhood immunization schedules has reduced 99% of invasive Hib disease to low levels ( nymph -> adult - ticks require blood meals between stages - no adult to egg transmission: ticks must acquire B. burgdorferi - mice, squirrels and birds can carry B. burgdorferi - majority of human infections come from nymphs Lyme disease transmission - tick inserts a feeding tube with barbs - secretes a local anesthetic - transmission is not thought to occur during the first ~24h following a bite - If tick is taken off before first 24 hours, there should be no infection - transmission increases >24h - nymphs normally transmit the disease - tick sucks blood slowly for several days - ticks appear grey when engorged Tick removal - use fine tipped tweezers - grasp the tick as close to the skin as possible - steadily pull upwards - thoroughly wash the area - keep the tick (to send for testing if necessary) - do not squish the tick body - Blood splatter can cause infection - do not burn the tick off - Because it stresses the tick out - do not apply petroleum jelly - Can be hazardous Borrelia burgdorferi  - following initial infection, B. burgdorferi must cause disseminated and persistent infection to propagate through its life cycle - hematogenous dissemination is a central event in the development of Lyme Borreliosis - Causes a bullseye rash upon infection (characteristic symptom) - Many people cannot see the bullseye rash - Caused by bacteria spreading through skin - compounds in tick saliva are thought to inhibit Dendritic cell function on multiple levels - decreased phagocytosis - decreased maturation - decreased inflammatory mediators - decreased antigen presentation - Tick secretes chemicals that prevents phagocytosis of bacteria - B. burgdorferi contain periplasmic flagella called axial filaments - Internalized but also sticks out of the bacteria - axial filaments wrap around cells to produce cork-screw shape - rotation of the axial filament causes the bacteria to move in a corkscrew like manner - promotes movement through extracellular matrix of host tissues and invasion of vasculature - B. burgdorferi contain an unusual outer membrane - no LPS despite being gram negative - many surface expressed lipoproteins that can act as adhesins - Adhesins are used to attach to the endothelial layer - escape from the vasculature requires adhesion to slow down B. burgdorferi - Bacteria requires flagella to pass through the endothelial layer of blood vessels - repetitive motility required to invade endothelium - Can also cause mild meningitis if it affects nervous system - B. burgdorferi has an unusual genetic structure - ‘linear’ chromosome - multiple plasmids – some linear and some circular - plasmids are required for infection but are variable from strain to strain - limited metabolic capability - Due to small genome - Requires mice/human to proliferate - Plasmids carry virulence factors - following initial infection, B. burgdorferi must cause disseminated and persistent infection to propagate through its life cycle - hematogenous dissemination is a central event in the development of Lyme Borreliosis Early localized stage - most common symptom is erythema migrans – painless 'Bulls-eye rash’ - ~25% of patients do not have a rash - They might have it but it could go unnoticed - occurs ~1-2 weeks after tick bite - groin, axilla, waist, back, legs, (head and neck in children)  - rash will expand and if untreated can reach >12 inches diameter - flu-like symptoms including fever, chills, fatigue, body aches - Not descriptive of the disease Early disseminated stage - occurs in untreated patients - multiple rashes would indicate dissemination of B. burgdorferi - pain and swelling of large joints - heart palpitations – interference with heart electrical signals - If bacteria goes to the heart - meningitis - severe headaches and neck stiffness - Bell's (facial) palsy – loss of muscle tone on one or both sides of the face Traversing the BBB - B. burgdorferi is though to use paracellular travel Late disseminated stage (months to years) - can cause serious long-term disability - response to antibiotics takes longer - muscle pain - arthritis - severe pain and swelling in large joints - ~5% of patients can develop neurological problems - shooting pains - Numbness - tingling in hands and feet - memory Post treatment lyme disease syndrome - 10-20% experience symptoms following treatment with antibiotics - cause is unknown - Hypothesized that your immune system is fighting antigens that look like the bacteria - lingering symptoms including - fatigue, muscle and joint pain, cognitive defects, sleep disturbances - may involve an autoimmune response or possibly persistent infection - most patients recover after a number of months - long-term antibiotics are not thought to help Lyme disease in Canada - Ticks continue to expand - migratory birds - warmer climate - Number of cases seems to have gone up - Could be because the disease is more known now Prevention  - avoid wooded areas endemic with Lyme disease - stay on paths, avoid low lying brush and long grass - wear long pants (tucked into socks) and long-sleeved shirts - light coloured clothing (to easily spot ticks) - repellants (containing DEET) - check for ticks and remove them Diagnosis - erythema migrans and other 'typical' symptoms - tick bite or reason to suspect tick exposure - anti-B. burgdorferi antibody tests (no 'Gold-standard') - detect antibodies to a laboratory strain of B. burgdorferi - false negatives often due to early testing (no antibody response yet) and genetic diversity of B. burgdorferi - sensitivity is somewhat controversial; not sensitive enough - submission of tick for testing (if you have it) to the National Microbiology Laboratory Treatment - if bitten by a black legged tick, watch for a rash (30 days) and be aware of symptoms - Bullseye rash - patients when diagnosed early will recover following antibiotic treatment - 2 - 4 week course of an antibiotic - without treatment: can lead to joint, heart, nervous system problems - ~10-20% of patients, typically with a late diagnosis, have post-treatment Lyme disease syndrome Chronic lyme disease - very controversial - some think this is due to chronic infection by B.burgdorferi - No lab confirmation - can be diagnosed without evidence of prior Lyme Disease - persistant symptoms including fatigue, headaches, sleep disturbances, cognitive dysfunction and other neurological problems - long-term antimicrobial therapy is not helpful – demonstrated by 4 clinical trials - Because it is not caused by B. burgdorferi - Most physicians do not believe this is real - previously, incidence of ~30,000 cases/year in the US and thought to be over-diagnosed - 3 CDC studies indicated that at least 300,000 cases/year in the US - most common in children (5-9 years old), and in women - case definition may have been too narrow - many symptoms in "chronic Lyme disease" not properly recognized - commercial tests probably not sensitive enough for B. burgdorferi - treatment may be too restrictive, too short Lyme disease vaccine  - LYMErix – based on an outer membrane protein - human trial (11,000 adults) showed it to be 75% effective - ‘should be considered’ for those in high risk areas (not ’recommended’) - claims (and lawsuits) that the vaccine caused arthritis – no evidence - Side effects - pulled from the market in 2002 due to ‘lack of demand’ – only now approved for dogs - Streptococcus classification - Inside this genus there are at least 100 different species - Streptococci are clinically divided into three major categories: - Α-hemolytic. - β-hemolytic. - γ-hemolytic. - This dividing is based on hemolytic capability - Alpha: partial hemolysis - Beta: complete hemolysis - Gamma: no hemolysis - Streptococcus pyogenes - Gram-positive, non-motile, non-spore-forming, and spherical cells, typically arranged in chains or pairs. - Circle chapped cells in a chain - Disseminated into the environment and/or colonizing humans and animals. - Recently , an increase of cases of zoonotic and nosocomial infections caused by different species of Streptococcus have been reported. - Also, call Group A strep (GAS) - Human-specific pathogen. - Encapsulated and β-Hemolytic. - Non-invasive or invasive infections - Non-invasive: an area where bacteria usually reside (skin, GI tract, etc) - Invasive: area that is usually sterile (blood, kidney, liver) Transmission and epidemiology - Transmission can occur through airborne droplets, surfaces contaminated with bacteria and skin contact. - Airborne droplets: invasive - Skin contact: non invasive -  - Annually, 616 millions of non-invasive and 1.8 millions of invasive cases are diagnosed around the world - Generating more than 500,000 deaths each year. - Ranked 5th among the most deadly infectious diseases in the world Non-invasive infections - Streptococcal pharyngitis or strep throat - Most common in children aged 5 to 15 years. - Symptoms: - sore throat, pain with swallowing, fever, enlarged tonsils, red spots on the roof of the mouth. - Diagnosis: - throat swab, which can be analyzed by a rapid strep test that reports results within about 15 minutes or bacteria culture. - Can also send culture to a lab for culturing - Impetigo - Common infection and highly contagious. - It is most prevalent in children aged 2-5 years old but can occur at any age. - Symptoms: - Erythematous plaques with a yellow crust, itchy or painful. - Plaques can occur anywhere on the body, but most common on face near mouth - Scarlet fever (scarlatina) - Rash associated with bacterial pharyngitis. - S. pyogenes exotoxin (superantigens) are mainly responsible for the manifestations. - Symptoms: - sore throat, fever, a red rash with a sandpaper-like feel; and a whitish coating on the surface of the tongue (strawberry-like). - Body wide red rash and white spots on tongue Invasive infections - Meningitis - Symptoms: - headache, neck stiffness, fever, and an altered mental status. - Diagnosis: - positive culture or bacterial DNA of cerebrospinal fluid. - Only way to be sure is to take cerebrospinal fluid and detect bacterial DNA - This disease has a mortality rate of 20%. - Recent 21-fold increase in cases generated by GAS in adults. - Necrotizing fasciitis (flesh eating bacteria)  - Necrotizing fasciitis is a subset of aggressive skin and soft tissue infection that causes necrosis of the muscle fascia and subcutaneous tissues. - Occur post-surgery, invasive procedures, or even a minor procedure. - These patients are extremely ill and should be transferred immediately to the intensive care unit. - Streptococcal toxic shock syndrome - It manifests with rapid progression and has the potential to culminate in shock, multi-organ failure, and with an acute-elevated mortality. - At this point, streptococcus is spread through the blood, producing toxins and virulence factors - Caused by superantigens which overstimulate the immune system which leads to extreme inflammation that can be life threatening Strep post-infection sequelae - Multiple infections with different or the same bacteria can cause an autoimmune response that persists even when the patient is not infected - Acute rheumatic fever -> rheumatic heart disease Virulence factors - Makes more than 30-40 virulence factors - Adherence - Fibronectin binding proteins (FBPs) - M protein - Capsule - Exotoxins - SLO and SLS: used for pore formation and cell death - Streptococcal pyrogenic exotoxins (superantigens): excessively activate the immune system - Immune modulation - Deoxyribonucleases (DNases): degrade NETs from neutrophils - IgG degrading enzyme (IdeS): cleaves antibodies - Exoenzyme - SpeB: cleavage of a wide range of host and bacterial proteins GAS pathogenesis 1. Adhesion: adheres to the host cells using the M protein and FBPs 2. Invasion: starts releasing virulence factors like SLO and SLS and superantigens to kill cells and multiply 3. Immune evasion: uses DNases to degrade NETs and the M protein to mimic host proteins - Also uses hemolysis to destroy RBCs and uses fragments of the dead cells to hide from immune cells in the blood 4. Dissemination: rapidly multiplies and spreads to other parts of the body M protein  - Virulence factor related to adhesion and anti phagocytosis. - This protein has been used to seroclassify this species. - There are >250 different serotypes of M protein within S. pyogenes Superantigens - There are at least 16 superantigens in S. pyogenes. - Molecules linked to the activation of the T-cells leading to cytokine storms. - This can cause toxic shock syndrome - Links TCRs and MHC-II receptors together, forcefully producing a stronger response - Superantigens are responsible for the establishment of infections and scarlet fever. Streptococcus pyogenes - In 2019 the incidence of scarlet fever and invasive infections increased 21-fold. - These cases were generated by a new sublineage identified as M1UK. - It has 27 chromosomal mutations. (27 changes in nucleotides) - Characterized by a 10-fold increase in the SpeA superantigen. Streptococcal DNases - Degrades NETs Treatment - 6- to 10-day course of amoxicillin (B-lactams) is the mainstay. - Patients with amoxicillin hypersensitivity (rash) requiring antibiotics should receive 10 days of clindamycin, Erythromycin, or clarithromycin. - B-lactams - There are no confirmed reports of B-lactams resistance in S. pyogenes Vaccines - There is no available vaccine against GAS. - There are currently eight candidates in medical trials. - S. pyogenes vaccines, including the potential for vaccine-induced Rheumatic Heart Disease due to auto-antibodies. - Some proteins in S. pyogenes are very similar to the collagen on heart - The vaccine would cause the immune system to attack the heart collagen which could lead to rheumatic heart disease Rheumatic heart disease - Most commonly acquired heart disease. - The disease results from damage to heart valves (mitral or aortic). - This disease has an autoimmune-inflammatory nature. - Immune system attacks the collagen in mitral or aortic valves - 34 million people were estimated to be living with RHD. - No biomarkers or treatments; only treatment is replacement Humanized mouse model  - S. pyogenes is a human-specific pathogen. - Regular mice cannot be colonized intranasally. - Humanized mice possess a MHC class II molecule similar to that found in humans. - Take the proteins from S. pyogenes and add them to a MHC-II molecule to produce a similar effect - Allowing the infection to establish in mice. - Repeated exposure to the proteins leads to rheumatic heart disease in mice - Echocardiogram is used to view the heart - Heart of mouse beats 500-700 beats per minute - Smaller than the tip of finger - In a normal mouse, there is no mixing of oxygenated and deoxygenated blood - Inflamed valves cause backflow of blood which can cause oxygenated and deoxygenated blood to mix, which causes heart disease Parasitology introduction What is a parasite? - Originates from the greek word parasitos (para = on/at/beside; sitos = food) - Broadly defined, a parasite is any organism that derives metabolic benefits by living on or inside a host of a different species - Must be 2 different species - Include animals, plants, fungi, bacteria, and viruses, which live as host-dependent guests - Harm is hard to quantify in biology, left out of definition - We mainly look at a specific subset of eukaryotes Organization of parasites - Protozoa - Proto = first; zoa = animals - Believed to be some of the first animals - Single celled eukaryotes - Metazoa - Evolved after protozoa - 2 types: - Helminths (worms) - Arthropods (insects) Parasitology: study of: parasite, host, relationship between both Advantages and disadvantages of being a parasite:  ADVANTAGES DISADVANTAGES Extreme host specificity can increase vulnerability to extinction Once host located, no need for further - Host needs to be alive searching - Some parasites can survive without a host for a short period of time, but not forever Food permanently available - Host does all the work Limited requirement for complicated food Must locate at optimal site on/in host to capturing mechanisms ensure food/survival Reduced need for food processing Protection from environmental extremes Must adapt to host’s internal physiological - Host provides protection environment (internal parasites only) Protection from predators and diseases Must overcome host’s immune defenses Reduced need for dispersal because the Spread limited by host’s geographic range host (+ vector) carries the parasite. Can devote larger proportion of energy Transmission can be extremely risky, and intake to reproductive output than a most offspring die before establishing in a free‐living organism new host - Requires a balance: antagonistic relationship Types of parasites - Facultative parasites: free living organisms that can turn into a parasite - Obligate parasites: cannot survive without a host - Endoparasites: lives inside body of host - Ectoparasites: lives on the surface of host body Parasitic protozoa - Over 200,000 species of protozoa have been described so far; 35,000 currently living - Many have been discovered in fossils - All protozoans are eukaryotic single-celled organisms - Free-living species occupy every conceivable ecological niche - 10,000 species have adapted for life as parasites - ~70 different protozoan parasitic species have been isolated from humans  - Parasitic protozoans infect a wide spectrum of vertebrate and invertebrate life Classification - Conventional classifications were according to mechanism of motility: - Flagellates (Mastigophora): have flagella used for motility - Amoeboids (Sarcodina): uses protoplasm to move - Apicomplexans (Sporozoa): move by gliding - Ciliates (Ciliophora): fine hair-like structure that moves - Modern classification schemes generally consider motility together with metabolism and DNA genotyping Mechanisms of entry Oral - Giardia duodenalis Sexual: good for narrow temperature range - Trichomonas vaginalis Inhalation: - Toxoplasma gondii (specific to cats) Direct contact: - Trypanosoma cruzi - Vector: defecates on skin and spread on mucosa of eye Arthropod vectors: - Plasmodium falciparum - Uses mosquito to go from host to host Parasite hosts - Parasites can have multiple hosts - Definitive: - The host in which the parasite reaches sexual maturity and undergoes reproduction (usually sexual). - For parasites that do not reproduce sexually, the definitive host is often the organism most crucial for completing the parasite’s life cycle. - Intermediate: - The host in which the parasite undergoes asexual reproduction or develops into its next stage but does not reach sexual maturity. - Reservoir: - A host that harbours the parasite and serves as a source of infection for other hosts (may be asymptomatic). Division and reproduction - Asexual: mitosis or binary fission - Sexual - Both - Monoxenous: 1 host  - Can undergo sexual or asexual reproduction - Diheteroxenous: 2 hosts - Sexual reproduction occurs in the final host - Triheteroxenous: 3 hosts - Generally more evolved Survival and pathogenesis - Antigenic variation: expresses different surface proteins each time to hide from immune system - Molecular mimicry: having different molecules that are similar to host proteins - Immune modulation: suppress or modulate the immune system - Intracellular habitation: gets inside cells to hide - Encapsulation: in a cyst/capsid; also provides protection from the environment - Protease secretion: enzymes that can cleave host proteins - Thermal tolerance: adaptive to temperatures - Toxin production: some metabolites are toxic to host - Nutrient deprivation: takes resources from host Antoni Van Leeuwenhoek discovered parasites for the first time under a microscope - Giardia cysts and giardia trophozoite most likely according to the description Giardia duodenalis - Synonyms: G. intestinalis or G. lamblia - Affects humans - Most prevalent protozoan human intestinal pathogen - Binucleated protozoan that inhabits the upper small intestine of vertebrate hosts - Both nuclei are transcriptionally active which is very unusual - Obligate parasite with a monoxenous life cycle consisting of two stages, the trophozoite and cyst - 1 host (humans) - Aerotolerant anaerobe (no mitochondria) - Transmitted to hosts through the fecal-oral route Giardia trophozoite - Adapted for survival within small intestine - 8 μm by 12 to 15 μm in size - Binucleate, both transcriptionally active - diploid - Four pairs of flagella - 8 flagella in total - Adhesive disc - Used to attach to the small intestine - Mitosomes - Contains iron sulfate and supports anaerobic respiration  - Originally thought to be primitive, but it does have genes for mitochondria that have been modified (coded for in the nuclear genome) - Basal bodies - Supports flagella Cyst - Environmentally stable - Has a cyst wall which provides protection from the environment - 5 μm by 7 to 10 μm in size - Tetranucleate (each nuclei is diploid) - Facilitates transmission - Metabolic rate is only 10% to 20% that of the trophozoite - Persists in environment to increase chance of uptake G. duodenalis life cycle 1. Cysts get ingested from contaminated water, food, or hands 2. Once the cyst reaches the small intestine, it undergoes excystation and becomes a trophozoite 3. Trophozoites attach to villi of small intestine and begin to multiply 4. Trophozoites rapidly reproduce asexually in the gut 5. Trophozoites are passed through stool, but they do not survive past the colon 6. Some of the trophozoites undergo encystation to turn into cysts and they survive after being passed through stool 7. The cysts from the stool remain in the soil until they are taken up by another host - Giardia can be observed in gerbils (different than the ones that infect humans) Epidemiology and pathogenesis - G. duodenalis is distributed worldwide - Cysts survive longer in cool, moist environments - Most parasites prefer warmer climates; this is an exception - Trophozoites adhere tightly to the small intestinal mucosa resulting in atrophy and flattening of the villi - Beavers are major reservoir hosts and often responsible for contaminating public drinking water (beaver fever) - Proteases and Secreted Substances - Cysteine proteases - Excretory secretory products (ESPs) - Membrane and Surface Proteins - To ensure that they survive in the environment - Variant-specific proteins (VSPs) - Attachment to villi can cause the villi to deteriorate and become inefficient at absorbing nutrients from food - Produces a similar effect to Crohn’s disease Diagnosis and treatment  - “Gold standard” for clinical diagnosis consists of microscopy of a fecal sample - Mostly used for pets (canines) - Antigen-capture ELISA or direct fluorescent antibody test (DFA) - Nucleic Acid Amplification Tests (NAATs) - PCR - Nitroimidazoles (e.g., metronidazole) are primary drugs used for treatment - Antibacterials that target anaerobes - Produces ROS in anaerobes and damages DNA - Does not affect host cells (aerobic) - We do not have enough redox potential to generate ROS from the drug; anaerobes have a much higher redox potential which reduces the drug into the active form which does damage to the DNA Protozoans I Malaria - The term malaria originates from Medieval Italian: mala aria or "bad air” - Before it was thought that the disease was caused by breathing in “bad air” - Passed from one human to another by the bite of an infected mosquito - Primarily affects children under the age of 5 - Numbers increased during covid because resources were not being sent Plasmodium parasites - Plasmodium spp. are members of the phylum Apicomplexa and responsible for causing malaria - Cannot survive independently as a free-living organism; rely entirely on host cells for reproduction and survival - Obligate parasite - Lifestyle requires two different hosts: - Vertebrate host (humans, bird, reptiles) for liver/blood stages - Different plasmodium species affect different hosts - Arthropod vector (mosquitoes) for sexual reproduction - Diheteroxenous (2 hosts) Plasmodium parasites in humans - There are more than 100 species of Plasmodium, which can infect many animal species such as reptiles, birds, and various mammals - Four species of Plasmodium have long been recognized to infect humans in nature: - P. falciparum  - Most severe infection (most deadly) - Tertian cycle: fever every 48 hours - P. vivax - Found mostly in asia and latin america - Tertian cycle: fever every 48 hours - P. ovale - Found in west africa - Tertian cycle: fever every 48 hours - P. malariae - Chronic illness; long term illness - Has a different life cycle: quartian cycle: every 72 hours - P. vivax and P. ovale are very similar but differ in how they infect the host - There is one species that naturally infects macaques which has recently been recognized to be a cause of zoonotic malaria in humans: - P. knowlesi - Can move between macaques and humans - Short cycle: every 24 hours Anopheles mosquito - Human malaria is transmitted only by females of the genus Anopheles - Of the approximately 430 Anopheles spp., only 30-40 transmit malaria (are vectors) in nature - Female mosquitoes take blood meals for egg production, and these blood meals are the link between the human and the mosquito hosts in the parasite life cycle - Females require blood for egg production; contains proteins and lipids necessary to create eggs - Males only eat sugar to sustain themselves Anatomy of female mosquito - Anterior midgut junction: specialized organ that separates blood meal and sugar meal - Dorsal diverticulum: digestion of sugar meal - Proboscis: used to take blood meal - Salivary glands: chemicals that get injected host; blood thinner - Crop: storage area for sugar - Stomach: blood meal goes to the stomach - Ovaries: reproduction - Malpighian tubule: similar function to kidneys - Anus: excretion of waste products - Hemolymph: circulatory fluid in invertebrates; functions like blood, but the circulatory system is open, all the organs gets bathed in hemolymph P falciparum life cycle (in mosquito)  1. Gametocytes in peripheral blood (of humans) - Gametocytes are the sexual form of the parasite - Macrogametocyte: female eggs - Microgametocyte: male sperm 2. Gametocytes are ingested by female mosquito during blood meal 3. Gametes form in the stomach of the mosquito - Sperm come out of microgametocytes through exflagellation and fertilize microgametocytes (eggs) to form a zygote (diploid) 4. zygote turns into an ookinete (tetraploid) in the wall of the stomach which is next to the hemolymph and turns into an oocyst 5. Thousands of sporozoites are formed through sporogony and released into the hemolymph - Sporogony = specialized form of mitosis 6. Sporozoites travel to salivary glands and stay in the ducts until the mosquito takes another blood meal - Remain in the ducts until they are injected with the saliva - Mosquito is not really bothered by the parasite because a mosquito’s life cycle is very short; mainly focused on reproducing Summary: Gametes (haploid) -> Zygote (diploid) -> Ookinete (tetraploid) -> oocyst (haploid) -> sporozoite (haploid) - Ookinetes are the motile form - Sporozoites are the active form that can infect humans P. falciparum life cycle (in humans) 1. Sporozoites enter bloodstream when a mosquito bites a human 2. They must hide from the immune system, and the only cells they can infect are hepatocytes in the liver - Migrate to the liver 3. Once a sporozoite enters the liver cell, it turns into a cryptozoite and multiplies through schizogony which is similar to mitosis, but there is no division of cytoplasm (no cytokinesis) - Forms a schizont which contains a large mass of cryptozoites that are all stuck together 4. Liver cell ruptures once the schizont is large enough, and all the cryptozoites undergo cytokinesis at the same time and turn into merozoites which can infect red blood cells 5. Merozoites infect red blood cells and form schizonts in the vacuoles of RBCs - Once they enter the red blood cells, they turn into trophozoites (ring form) - This changes the way the RBCs look by expressing sticky proteins 6. Trophozoites rapidly multiply and produce more merozoites and 5-10% of them turn into gametocytes - Mechanism by which merozoites release from RBCs is not well understood; they probably rupture the cell  - Merozoites differentiate into gametocytes either by epigenetic changes or genetic changes - Called gamogony 7. When another mosquito comes to take a blood meal, it takes up gametocytes and merozoites - Merozoites have no effect on the mosquito - Humans are the intermediate host - Mosquitoes are the definitive host - Because it sexually reproduces in the mosquito’s stomach Symptoms and diagnosis - Quickest way is to do a blood smear: tells you which species it is - This requires knowledge of what the species looks like - PCR - Takes a long time + can be expensive - Symptoms - Headache - Fever - Fatigue - Pain - Chills - Sweating - Dry cough - Enlarged spleen - nausea/vomiting Other modes of transmission - Direct contact with infected blood - Merozoites in the blood can infect your blood directly - Vertical transmission: infected mother transmits the parasite to the fetus - Also if the infant becomes infected within 7 days of birth = vertical Survival and pathogenesis Host invasion - Injected into bloodstream by Anopheles mosquito - Sporozoite entry into hepatocytes - Merozoite entry into red blood cells Immune invasion - Antigenic variation: expresses different proteins at different times to trick immune system - Intracellular hiding: hides inside of cells Toxin production: hemozoin: digests hemoglobin inside of RBCs and releases the heme group, which is toxic to the cells in the free form (also toxic to the parasite) - Has an enzyme that stacks the heme into a crystal (hemozoin) which is toxic to cell - There is fossil records of plasmodium being on Earth for a very long time Treatment - Based on: - Infecting Plasmodium species - Geographical location tells you a lot - Blood smear - Clinical status of the patient - Pregnant, HIV, etc - Expected drug susceptibility of the infecting parasite as determined by the geographic area where the infection was acquired - Can be used to determine the species - Previous use of antimalarials, including those taken for malaria chemoprophylaxis - 2 common drugs: - Artemisinin: acts as a damaging free radical and kills parasite cells - Chloroquine: inhibits the enzyme that makes hemozoin, which is toxic to the parasite (and the cells, but the parasite is more affected) - Some parasites have been shown to develop resistance to this drug - Chemoprophylaxis: similar to a vaccine, used in pregnant women and infants to prevent the disease Incidence of malaria - The density of suitable habitats for mosquito vector breeding - More mosquitoes = more infections - The prevalence of infected humans that mosquitoes can feed on - More infected humans = more infections - The susceptibility of humans bitten by an infected mosquito - More bites = more infections - Isolate infected mosquitoes - North America has almost no malaria cases - Most are from people who travel overseas - People who live in endemic regions have adapted immune systems that allow them to survive the disease Why is the parasite hard to eradicate?  - Parasite is good at evading immune system - Has worked in some areas but not completely - Mosquitoes are vital to the ecosystem - Coevolutionary antagonism: sickle cell trait and malaria - People with sickle cell trait has a natural defense against malaria Sickle cell trait and malaria coevolution - Sickle cell gene is caused by a single amino acid mutation in the beta chain of the hemoglobin gene - Inheritance of the mutated gene from both parents leads to the sickle cell disease - Individuals who are heterozygous carriers for sickle cell gene have some protective advantage against malaria - 1 normal gene + 1 mutated - Frequency of sickle cell carriers are high in malaria-endemic areas - HbSS gene does not provide immunity, it makes the parasite less successful - Beta subunit is mutated; forms fibres in the RBCs - Affects parasite by preventing entry into RBC and using hemoglobin - Fibres prevent digestion - Mutation converts glutamate (positive charge) -> valine (non-polar) Prevention - Vector Control + Personal Protection - Case management - Insecticide-treated nets - Mosquitoes tend to bite people indoors - Indoor residual spraying - Prophylaxis treatment (pregnant women & infants) - Vaccine for children - RTS,S/AS01 (Mosquirix) - R21/Matrix-M - Similar mechanisms: - Target the sporozoite (prevents liver infection) - Not very available in malaria endemic regions Helminths (worms) Parasitic metazoa - Metazoan parasites are multicellular organisms expressing both ecto- and endo parasitic lifestyles - Macroscopic, multicellular organisms - Can vary in size from millimeters to meters in length (quite long) - Includes helminths (worms) and arthropods (insects and arachnids) Classification of helminths  - Nematodes: unsegmented roundworms - Similar to earthworm shape; completely unrelated though - Cestodes: segmented flatworms - Tapeworms, not smooth - Trematodes: non-segmented flatworms - Similar to a hybrid between the two previous types Mechanisms of entry - Fecal-oral: eggs/larva are ingested - Transdermal: some can invade tissue by breaching skin barrier - Vector borne: through animals - Predator-prey: transmitted by prey Niche selection - Infects a variety of organs, depends on the species Mechanisms of survival - Incorporation of host serum proteins on surface: hide from immune response - Displays host proteins to hide from immune cells - Inhibition of the complement system - Secretion of anti-inflammatory molecules - Avoiding direct contact with host tissue (living in the lumen of the small intestine) - Inside the body, but considered external - Pausing life cycle when host develops resistance (dormant stage) Ascariasis (name of disease) - Ascaris lumbricoides are very large nematodes that parasitize the small intestine - An estimated 807 million – 1.2 billion people in the world are infected - Parasitic infection occurs through ingestion of eggs via fecal contamination of soil, foodstuffs, and/or water supplies - Human waste is commonly used as fertilizer in many parts of the world - Obligate parasite - monoxidous Life cycle: 1. Eggs are ingested by host 2. Larvae hatch in the small intestine, enter the bloodstream and go to the liver - Pass through the intestinal wall and migrate to the liver - Use liver as a food source 3. Larvae migrate to heart, where they can reach the lung capillaries 4. Larvae enter the alveoli of the lungs and grow there until they are forced out by pressure system 5. Larvae go up the trachea where they are swallowed and go back to the small intestine  6. Adults mature in small intestine and eggs are released in the feces where they can be taken up by another host - The disease can be low burden or high burden - Can cause bowel obstruction in some cases Symptoms - People rarely show any symptoms, and if they do, usually mild abdominal discomfort - Problem occurs when disease is untreated and/or there are other concurrent factors - Ascariasis & malaria - Common in endemic regions with malaria - Cyclic fever causes worms to escape via moving up (through the throat) or through the colon - Can cause choking Anthelmintic treatment - Anthelmintic refers to a substance or drug that is used to treat infections caused by helminths - Mechanisms of action include killing the parasites and/or expelling them from the host's body - Albendazole - binds to β-tubulin and disrupts microtubules - Same family as giardia drug - Ivermectin - binds with high affinity to glutamate-gated chloride channels found in nerve and muscle cells of invertebrates, including nematodes and arthropods - Also considered a cancer drug - Worms fight against peristalsis, if they cannot move, they will be excreted Taeniasis - Taenia saginata species (beef tapeworm) are very large cestodes that parasitize the small intestine - Cows become infected after feeding in areas that are contaminated with Taenia eggs from human feces - Humans can become infected with tapeworms when they eat raw or undercooked beef - Tapeworms are hermaphrodites: contain both male and female organs in the segments - Obligate parasite - Diheteroxidous (2 hosts: cows and humans) - Humans: vital host - Cow: intermediate host Life cycle 1. Cysticerci (larval stage encapsulated in a cyst) are ingested with raw or undercooked beef 2. Cysticerci degrades to release larva in the stomach (of humans)  3. Worms mature and live in the small intestine - Usually there is only 1 worm per person due to competition - Uses a scolex (sucker with 4 tubes) to adhere to the small intestine 4. Produce proglottids (segments that contain eggs) which are passed in feces - Adults can grow up to 10 meters in length - Gravid proglottids break off of the main body and release into feces 5. Cows ingest embryonated eggs, which turn into oncospheres (larval stage) which migrate to muscle tissues to develop into cysticerci Symptoms and treatments - Most people with tapeworm infections have mild symptoms or no symptoms - White spots in feces -> proglottids - Treated with anthelmintic medication praziquantel - Hypothesized to disrupt calcium homeostasis resulting in uncontrolled calcium ion influx - Causes paralysis in the worm, and leads to it being excreted Neglected tropical diseases - Diseases that there aren’t enough resources to treat - Unfair for people living with these diseases, despite them being a small minority Anti parasitic measures - Improve the efficacy, cost-effectiveness, ecological soundness and sustainability of disease-vector control - Mosquitoes breed in wet marshlands - Bites are more likely to occur indoors - Some mosquitoes prefer animal hosts - Deforestation can change the habitat of mosquitoes - More biting at night - Reduction of areas where vectors breed - Access to bed nets and/or other physical barriers against mosquitos - Treatment of areas with pesticides - Introduction of genetically modified mosquitoes Other problems - Complex, difficult, or impossible to solve problems that affect people, communities, and businesses. - Incomplete, contradictory, and changing requirements - Lack of clarity - Real-world constraints - Unknown number of potential solutions Mycology introduction  - Fungi evolved long before us and plants evolved even before them - Started out as small plants will little leaves and short stems - Prototaxites: believed to be a fungi that existed millions of years ago - Covered earth and grew as tall as trees - Allowed for other species to grow - Armillaria: filamentous fungi; one of the largest organisms on Earth - Fungi ruled the Earth long ago - Fungi are somewhere between plants and animals but closer to animals Plants vs Fungi Plant Cell Fungal Cell Most fungi have a cell wall made of chitin Plants have a cell wall made of cellulose - Made of B(1-4) linked - Made of B(1-4) linked D-glucose N-acetylglucosamine Generally, possess one nucleus per cell May be uninucleated or multinucleated Autotrophs (can produce energy from the Heterotrophs (need to get energy from other sun) organisms) Membranes contain ergosterol Membranes contain phytosterols (animals have cholesterol) Store food as starch in granules Store food as glycogen in granules - Amylose a(1-4) linked glucose (similar to animals) (unbranched) - a(1-4) + a(1-6) - Amylopectin a(1-4) + a(1-6) linked - Similar to starch but more branched glucose (branched) Classification of fungi  - Spore Formation - Structures are very different at different parts of their lives - Fungal Genomics - Modes of Nutrition (heterotrophs) - Saprophytic – The fungi obtain their nutrition by feeding on dead organic substances - Example: aspergillus/penicillum - Parasitic – The fungi obtain their nutrition by living on other living organisms (plants or animals) and absorb nutrients from their host - Example: puccinia; rust type fungi that causes blights in plants - Symbiotic – These fungi live by having an interdependent relationship with other species in which both are mutually benefited - Lichens - Example: wolf lichen; used to keep wolves away (pigment is toxic) - Mycorrhiza - Filamentous fungi: can produce fruit bodies - Microfungi are the mold form - Yeast - Mostly mold and unicellular (not all of them) - Morphology can vary between life periods Fungal reproduction Asexual - common mode of reproduction in fungi - Fungi undergoing asexual reproduction are described as anamorphs - Asexual propagules are considered spores produced following mitosis - Piece of fungal unit that goes out into the environment Sexual - Fungi undergoing sexual reproduction are described as teleomorphs - Sexual propagules are produced by the fusion of two nuclei that then generally undergo meiosis - Fusion of 2 cells to become diploid - No male and female gametes (have different names) Parasexual - Involves genetic recombination without the requirement of specific sexual structures - Undergo genetic recombination without sexual structures Filamentous fungi - Fruitbody releases spores  - Spores germinate if conditions are favorable - Spores produce hyphae in the soil - Can mate in the soil between hyphae from different spores - Extremely diverse and various structures possible Vegetative growth - Fungi typically grow as filaments, termed hyphae(singular: hypha), which extend only at their extreme tips - Fungi exhibit apical growth in contrast to many other filamentous organisms - Fungal hyphae branch repeatedly behind their tips, giving rise to a network termed a mycelium - White threads in soil = mycelium - 2 types of hyphae: - Septate: separated nuclei - Coenocytic: no separation between nuclei or any organelles - Organelles can freely flow through the hyphae - Hyphae age as they grow; some part of the hyphae may die while another part is growing Aspergillus nidulans life cycle Sexual cycle: 1. Mating occurs between 2 hyphae branches (some fungi can do it between 2 branches of their own hyphae) - With the same species 2. Forms a cleistothecium (sexual structure) which contains ascospores stored inside of ascus - There are 8 ascospores inside each ascus - No male/female; 2 nuclei of different types fuse together making a diploid structure which undergoes meiosis (producing 4 progeny) and each of the progeny undergo mitosis once (in total producing 8 spores) - Hulle cells are on the cleistothecium to nurse the ascospores Asexual cycle: 1. Hyphae produces a conidiophore which produces conidium (spores) 2. The conidia can germinate in another location if the conditions are right Parasexual cycle: 1. Nuclei inside of the hyphae fuse together to create a heterodikaryon (N) 2. The hyphae becomes diploid 3. Undergoes haploidization to lose a set of chromosomes and return to a haploid - This is unregulated and can even cause the hyphae to die in some cases Yeast  - Approximately 1% of described fungi grow as single-celled yeasts (e.g., Saccharomyces cerevisiae) - Filamentous fungi are multicellular - Some species are dimorphic and can switch between a yeast phase and a hyphal phase in response to environmental conditions (pseudohyphae) - Fission yeast - Divide by binary fission; genetic material gets sent to poles and the cell splits - Budding yeast - Daughter cell buds off the parent cell - Pseudohyphae - Change their morphology and resembles a hyphae structure Saccharomyces cerevisiae life cycle Asexual: 1. Budding yeast; daughter cells bud off the parent cell and grow until they are big enough 2. Once the cell is big enough it detaches from parent cell Sexual: 1. ‘A’ and alpha mating types (no male and female) find each other and form shmoo cells - The cell release pheromones which they use to find each other - Cells can also switch their mating type from a to alpha or vice versa 2. The shmoo cells fuse together through nuclear fission 3. The nuclei of both cells fuses to make a zygote 4. Zygote can directly start budding (mitosis) and produce copies of itself before meiosis 5. The diploid zygotes form an ascus and undergo meiosis and sporogenesis 6. The ascus releases 4 ascospores - Nutrient deprivation helps toggle the switch between sexual and asexual reproduction - The cell senses a threshold Schizosaccharomyces pombe life cycle - Essentially the same as the the other fungi Sporulation - Dissemination - Reproduction - Allow fungus to move to new food source: spores can travel quite far - Allows fungi to survive periods of adversity - Dormant under unfavorable conditions - Introduce new genetic combinations into a population: sexual reproduction - Source of inocula for infection Benefits of fungi  - Nutrient Cycling - Nitrogen fixation - Carbon sequestering - Phosphorus - Carbon Cycling and Climate Regulation - Nutrition and Food Security - Human Health - Antibiotics - Anti inflammatory drugs - Environmental Protection - Certain organisms can eat oils/plastics - Sustainable Materials - Can be used to make materials Fungi as food - Mushrooms have been used for food, medicinal purposes, as hallucinogenic agents in rituals, or as a means to start a fire (tinder species) - There are nearly a hundred species of fungi for which some kind of cultivation system is known—all of these cultivated species are saprophytes - Mushrooms have a delicate life cycle - Majority of mushrooms come from china - 1100 species of mushrooms eaten in more than 80 countries Yeast in food production - 2 main uses: - Baking - The most common yeast used in breadmaking is Saccharomyces cerevisiae - Feeds on sugars present in bread dough, producing carbon dioxide gas - Helps bread rise - Other ingredients affect speed of fermentation: - sugar and eggs speed it up - fats and salt slow it down - Ethanol is evaporated when the bread bakes - Making alcoholic beverages - Most fungi are obligate aerobes, but can function as anaerobes to produce ethanol - Several different yeasts are used in brewing beer - Ferment sugars present in malted barley to produce alcohol - Saccharomyces cerevisiae, is used to make ale-type beers and is known as a top-fermenting yeast - Bottom-fermenting yeasts, such as Saccharomyces pastorianus, are more commonly used to make lagers - Flavors depend on the type of yeast - Wine-making  - The alcohol in wine is formed by the fermentation of the sugars in grape juice often by Saccharomyces cerevisiae - You can isolate yeast from grape skin - Sparkling wine is made by adding further yeast to the wine when it is bottled—the carbon dioxide formed in this second fermentation is trapped as bubbles - Cheese making - Filamentous fungi are important in the manufacture and ripening of two types of cheese: - Blue-veined cheeses (e.g., Roquefort, Gorgonzola, Stilton, etc.) - Cheese is pierced with metal rods to allow fungi to grow inside - Soft-ripened cheeses (e.g., Brie, Camembert, Humboldt Fog, etc.) - Allow fungi to grow on the outside to create a rind - Mycoprotein - During the 1960s, it was predicted that by the 1980s there would be a shortage of protein-rich foods - Scientists and international aid agencies attempted to develop protein-rich foods from microbial biomass - A massive bioreactor was created where fungi could be continuously harvested - Developed of an entirely novel food product, termed Quorn™ mycoprotein - Quorn mycoprotein is produced commercially from chemostat cultures of mycelium from the filamentous fungus Fusarium Venenatum - Only around 3% of mushroom varieties are poisonous to humans Medical mycology Health risks posed by fungi - Fungal infections, or mycoses, are diseases caused by a fungus (yeast or mold) - Only about 200–300 fungi are reported to cause diseases of humans and other warm-blooded animals - Mycotoxins are toxic chemical compounds produced by certain fungi that grow on food crops and other organic materials - Airborne spores can trigger asthma, allergies, or cause occupational diseases Fungal virulence - Over a billion people are affected by fungal disease; approximately 1.5 million deaths are attributable to fungi annually - Transient exposure to fungi or fungal colonization occurs without the knowledge of the affected individual - Most fungi have low virulence - Difficult to diagnostically distinguish between presence and infection Classifying mycoses  - Fungal infections may be classified according to: - Site of infection - Superficial, cutaneous, subcutaneous, or systemic - Route of acquisition - Exogenous or endogenous - Type of virulence - Primary mycoses - Opportunistic mycoses Dermatophytic fungi - ​Most superficial and cutaneous mycoses are caused by dermatophytes - Most dermatophyte infections are caused by molds of the genera Trichophyton, Microsporum, and Epidermophyton - Infections are usually self‐limiting; generally, no cellular immune response - Can be treated relatively easily using topical antifungal drugs; in severe cases oral drugs may be administered - Athlete’s foot - Ringworm Opportunistic systemic mycoses - Opportunistic mycoses exploit the imbalance between the host and the pathogen that occurs in immunocompromised individuals - The two most common opportunistic fungal pathogens are yeast species belonging to the genus Candida and molds belonging to the genus Aspergillus Risk factors - HIV infection and AIDS - Solid‐organ transplantation - Anticancer chemotherapy - Granulocytopenia - Premature birth - Old age - Use of corticosteroids - Use of broad‐spectrum antibiotics - Central vascular catheters - Gastrointestinal surgery - Colonization with fungus Candidiasis - A large proportion of humans innocuously carry several Candida species on epithelial surfaces - Candida species most frequently associated with human infection is Candida albicans - Superficial candidiasis infections include: - Oropharyngeal candidiasis - Denture stomatitis - Vulvovaginal candidiasis - Chronic mucocutaneous candidiasis Virulence factors of candida - Adhesins - Dimorphism - Phenotypic switching - Extracellular hydrolases Invasive Candidiasis Symptoms and diagnosis - Examples of symptoms include fever and chills, low blood pressure, muscle aches, skin rash, weakness or fatigue, etc. - Diagnosis using: - selective media cultures - detection of anti‐Candida antibodies and Candidaantigens in blood samples - Epidemiology: DNA fingerprinting, microarrays, and PCR - Aspergillosis - Approximately 20 Aspergillus species have been associated with human infections - Aspergillus are saprophytic fungi ubiquitous in the environment - Aspergillus species most frequently associated with human infection is Aspergillus fumigatus Virulence factors of aspergillus - A. fumigatus conidia are dispersed very easily in air and routinely inhaled by humans - Immunocompetent individuals: conidia are detected and destroyed by alveolar macrophages - Immunocompromised individuals: spores can settle, germinate, and invade, ultimately leading to invasive aspergillosis - Thermal tolerance - Proteinase production - Gliotoxin production - Environmental stress resistance Invasive aspergillosis - Alveolar infection - Angioinvasion - Dissemination Symptoms and diagnosis - Invasive aspergillosis has a high mortality rate (>50%) - Symptoms are nonspecific, usually include fever, sometimes chest discomfort, and cough (sometimes with blood) - Gold standards of diagnosis include histopathological analysis and culture of biopsy and bronchoalveolar lavage fluid taken from the infected area of the lung Endemic systemic mycoses - Geographically restricted to certain regions - Histoplasmosis - Coccidioidomycosis - Blastomycosis - Paracoccidioidomycosis Antifungal agents - Plasma Membrane - Polyenes: binds directly to ergosterol - Azoles: inhibits 14-alpha demethylase - Cell Wall - Echinocandins: inhibition of B(1-3) glucan synthase - Nikkomycin: inhibition of chitin synthase - Nucleic Acid & Protein Synthesis - 5-fluorocytosine: interferes with nucleic acid synthesis by disrupting synthesis of thymidine and protein synthesis by integrating into RNA - Sordarin: interferes with protein synthesis by binding to fungal elongation factor 2 (