Summary microbiology History Antoni van Leeuwenhoek: described animalcules = protozoa and bacteria, first man that studied microbes. Linnaeus: developed the taxonomic system Aristotle: spontaneous generation Spallanzani: first one that tried to prove that spontaneous generation doesn’t exist Pasteur: proved that spontaneous generation doesn’t exist ® father of microbiology Koch: proved that diseases are caused by bacteria (anthrax and TB), Koch’s postulates - The cause of the disease must be found in every case of the disease and be absent in healthy people - Agents must be isolated and grown outside the host - When the agent is introduced in a healthy host it must cause the disease - The same agent must be found in the diseases experimental host Semmelweis: hygiene theory Jenner: first vaccine ® against smallpox (DNA virus) Cell structure and function Bacterial cells: - Glycocalyx: composed by polysaccharides and polypeptides, gelatinous sticky substance, two types ¨ Capsule: firmly attached to cell surface, prevents recognition by host + phagocytosis ¨ Slime layer: loosely attached to cell surface, allows cell to attach to surfaces ð Favor formation of biofilm - Flagellum: made by filament (helix made by flagellin), hook (where filament inserts), basal body (anchors hook and filament to wall and membrane); differences in flagellin allow the classification in serovars - Axial filament: observed in spirochete, located between inner and outer membrane, allows it to cross membranes - Fimbriae: adhere to one another and to surfaces, they’re a mechanism of virulence. With the capsule they allow the formation of the biofilm - Pili: transfer of DNA between cells (conjugation) - Cell wall: protection from osmotic forces, made by peptidoglycans (disaccharides connected by cross-bridges made by 4 aa) ¨ Gram +: thick cell wall containing teichoic acids and lipoteichoic acids; stain purple; mycobacterium ® cell wall contains mycolic acid (waxy lipid that protects from desiccation) ¨ Gram -: thin cell wall, membrane has an normal inner layer and the outer layer contains lipopolysaccharides ® O side chain recognized by antibodies (varies among strains); core domain; lipid A = major virulence factor, recognized by TLR4 - Cytoplasm: contains inclusions (contain lipids, starch, glycogen, compounds containing sulfur) - Endospores: dormant structure extremely resistant, contains dipicolinic acid and calcium; formed by Clostridium and Bacillus Eukaryotic cells: - Glycocalyx: anchors cells to one another ® cell-cell recognition and communication - Membrane rafts: groups of lipids and proteins, used by viruses to enter the cell 1 - Flagella: within the cytoplasmic membrane, made by globular tubulin, no hook Microscopy and staining Staining is important to increase the contrast; electron microscope ® staining with heavy metals. Staining: spreading over a slide ® desiccation ® fixation (with heat or chemicals) - Gram stain: I stain = crystal violet, fixer = chemical (mordant), counterstain for gram- = pink - Acid fast stain: fixed with heat, used for bacteria with waxy lipids in cell wall (Nocardia and Mycobacterium) - Endospore stain: fixed with heat, for Clostridium and Bacillus - Histological stain: gomorri methenamine silver ® fungi - Negative stain: to observe the capsule Microbial metabolism, nutrition and growth Growth requirements: - Source of carbon: CO2 ® autotrophs, organic source ® heterotrophs - Source of E: chemicals ® chemotrophs, light ® phototrophs - Source of electrons: organic molecules ® organotrophs, inorganic molecules ® lithotrophs Temperature (going from low to high): psychrophiles – mesophiles (human pathogens) – thermophiles hyperthermophiles. Biofilms: primary residence of microbes in nature, cause 70% of diseases; microbes secrete quorum sensing molecules that causes cells to change their structure and biochemistry, it may also regulate genes. Culturing: - Inoculum: a sample collected from a biopsy and introduced in the culture - Culture: the microorganisms that are cultured - Colony forming unit: progenitor of colony Methods to obtain pure cultures: - Streak plates: inoculum spread across a plate - Pour plates: dilution sample, then poured on a surface Culture media: - Nutrient agar: used to obtain a semi solid medium - Synthetic media: you know the exact composition, used to culture fastidious microbes - Complex media: undefined composition, usually add blood - Selective media: add compounds that allow the growth of specific microbes - Differential media: one component of the media is used in different ways by different species, ex blood (alpha, beta and gamma hemolysis) Measuring microbial reproduction: - Direct methods: ex membrane filtration ® estimate microbial population size - Indirect methods : ex turbidity ® measured with spectrophotometer Plasmids: circular molecules of DNA - Fertility ® instructions for conjugation - Resistance ® genes for resistance to antimicrobials and heavy metals - Bacteriocin ® genes for toxins (bacteriocins) ® kill competition - Virulence ® genes for structures, enzymes and toxins Genetic transfers in prokaryotes: - Transformation: DNA taken from environment - Transduction: DNA passed from one cell to another via virus - Conjugation: mediated by pili 2 Control of microbial growth Sterilization = destruction of all microbes in or on an object Disinfection = destruction of all microbes from non living tissues Asepsis = reduction number microbes (from patients) Degerming = mechanical removal of microbes from objects and tissues (hand washing) Sanitization = removal microbes to reach public health standards Antimicrobials: efficiency measured based on the number of microbes dying per minute (= microbial death rate, constant over time); mechanisms of action: - Alteration of cell walls and membranes ® cell dies due to osmotic pressure; enveloped viruses are the most susceptible because if you destroy the envelope they won’t be able to attach to cells anymore - Damage to proteins and nucleic acids Resistance: prions > endospores > mycobacterium > cysts of protozoa > gram- > non-enveloped viruses > gram+ > enveloped viruses. Germicide classification: - High level: oxidizing agents, kills all pathogens - Intermediate-level: alcohol, halogens, kills spores, cysts, viruses and bacteria - Low level: soaps and detergents, kill bacteria, fungi, protozoa and some viruses Levels of biosafety: 1. Soaps and disinfectants ® E. coli 2. Safety cabinets ® MRSA and hepatitis 3. Air filters, double door, negative pressure ® TB, anthracis, yellow fever 4. Separate building, sealed airlocks, multiple showers, vacuum rooms, filtration of air, water and pressure ® Ebola, smallpox Physical methods of microbial control: - Moist heat: more effective than dry heat ¨ Boiling: 100°C for 10 min/h ® not a sterilization method ¨ Autoclaving: boiling T increases as pressure increases, 121°C for >15min ¨ Sterilization: 140°C for 1-3s - Dry heat: for materials that can’t be sterilized with moist heat, 160°C for 2h - Refrigeration and freezing: stops the growth of most pathogens, except Listeria and Yersinia that reproduce in refrigerated food and blood respectively. Slow freezing is more effective than quick freezing. Freezing doesn’t kill microbes! - Electromagnetic radiations: gamma rays used to sterilize medical equipment, UV light used to eliminate microbes on the surface of objects (doesn’t even cross dust) - Filtration: membrane filters with pores with defined size, present in safety cabinets, OR Mechanism of antimicrobial action: based on selective toxicity ® targets something specific for a certain microbe. - Inhibition of cell wall synthesis: penicillin contains a beta-lactam ring that inhibits the formation of cross links to form peptidoglycans, targets microbes in log phase (growing phase). - Inhibition protein synthesis: targets ribosomes - Inhibition of specific metabolic pathways: ex sulfonamides inhibit synthesis folic acid Spectrum of action: - Narrow spectrum: ex penicillin - Broad spectrum: ex tetracyclin, can cause secondary infections and kills our microbiota Effectiveness: - Diffusion susceptibility test: measure the zone where there’s inhibition of growth - Minimum inhibitory concentration test: measure concentration needed to inhibit the growth 3 Routes of administration: - Intramuscular: fast increase in concentration - IV: longer increase in concentration (reach a plateau and maintain it) Therapeutic window = range of effective concentrations not toxic for the host Resistance: - Degradation drug: ex beta-lactamase degrades beta-lactam of penicillin - Alteration of target: ex absorbing folic acid from environment to bypass sulfonamide - Alter porins: drugs can’t enter the cell - Efflux pumps: pump out drugs - Biofilms: retard the diffusion of drugs Bacterial persistence = bacteria in resisting state are resistant to drugs that target the replicative state. This persistence occurs depending on environmental conditions. Bacteria in biofilm are in resting state. Resistance can be distinguished in: - Multiple drug resistance ® resistant to three or more antimicrobial agents - Cross resistance ® when drugs are similar in structure, the resistance to one drug can confer resistance to other similar drugs Characterizing and classifying prokaryotes Reproduction: - Binary fission: most common - Snapping division: typical of C. diphteriae, a portion of the cell wall remains ® form chains - Production of reproductive spores: specific for actinomycetes - Budding: cell doesn’t divide in two equal portions Archea: not a cause of disease, they lack true peptidoglycans, also membrane lipids are different; they can live in extreme conditions. They’re the major source of environmental methane. Listeria = contaminated milk and meat, survives in phagocyte. Lactobacillus = member of microbiota used for probiotics, not pathogenic. Staphylococcus, streptococcus and enterococcus = multi-drug resistance. Actinomycetes = made by branching filaments that resemble fungi, cause diseases in immunocompromised patients ® actinomyces (abscesses), nocardia (pneumonia, cutaneous and CNS diseases), Streptomyces (recycle nutrients in soil). Chlamydia: intracellular parasites, cause neonatal blindness, pneumonia, sexually transmitted lymphogranuloma venereum. Classification eukaryotes Some protozoa reproduce through schizogony: nucleus undergoes multiple mitosis forming schizont (polynucleate), after cytokinesis they release merozoites. Protozoa are not an accepted taxon, they’re grouped because they have similar characteristics. They can have 2 nuclei: macronucleus = contains several copies of genome, and micronucleus = involved in genetic recombination and sexual reproduction. Stages in life of protozoa: - Cysts: resting stage, it’s the infective form - Trophozoite: develops from cysts when it’s inside host, causes disease Fungi: cause diseases called mycoses, there are 4 true pathogens and the others are opportunistic, most are beneficial. The body is called thallus, it forms hyphae in molds (hyphae form the mycelium), it’s composed of small single cells in yeasts and dimorphic fungi have both forms. They all have means for asexual reproduction, most of them also reproduce sexually. Molds produce spores that can be called: sporangiospores (form inside a sac = sporangium), chlamydospores (form 4 inside hyphae) or conidia (form at tips of hyphae). Candida albicans produces long filaments called pseudohypha = several buds remaining attached ® invade tissues. Sexual reproduction (when there’s a limitation of nutrients): dikaryotic stage = two hyphae fuse, the nuclei don’t fuse, they form the dykarion; diploid stage = fusion of two nuclei then meiosis (possible genomic recombination ® confers advantages to resist the environment), haploid stage = meiosis produces spores that will develop into hyphae. Helminths (parasitic worms): either dioecious (male and female sex organs in two separate organisms) or monoecious (each worm has both sex organs). Three groups: - Cestodes: tapeworms, intestinal parasites, structure called strobili, formed by segments called proglottid (each one containing both female and male sex organs). At one extremity there’s the scolex (head) and in the other a gravid proglottid. - Trematodes: flukes - Nematodes: roundworms, parasites of almost all vertebrates Arthropod vectors: vectors that only carry pathogens are called carriers, vectors that function also as a host are called biological vectors. - Arachnida: ticks are the most important, ex vectors for Lyme, rickettsiosis - Insects: ¨ Fleas ® Yersinia pestis, intermediate host of tapeworms ¨ Lice ® typhus ¨ Flies ® Leishmania, sleeping sickness ¨ Mosquitoes ® malaria, yellow fever, dengue, filariasis, viral encephalitis ¨ Kissing bugs ® Chagas Viruses Extracellular state is called virion, made by capsid + nucleic acids (= nucleocapsid). Capsid and envelope confer protection. Most viruses are able to infect only one host. Capsid: protection + attachment to host cell, made by capsomeres, it confers the shape to the virus. Envelope: acquired from the host’s membrane when virus is released (ER membrane, plasma membrane or nuclear membrane). Viral replication is dependent on host’s organelles and enzymes, they’re different if the virus is infecting bacteria or humans. Bacteriophages: - Lytic replication: attachment to host is mediated by proteins on tail that bind to proteins on cell wall; lysozymes form a hole on the membrane ® injection genome; assembly virion; lysis host cell - Lysogenic replication: done by temperate/lysogenic phages, when they’re inside the chromosome they’re called prophages or inactive phages. The genome is integrated in chromosomes (lysogeny), afterwards there’s a phase called induction in which the genome detaches from chromosomes and enters the lytic cycle. Replication animal viruses: attachment to host is mediated by attachment molecules or glycoprotein spikes (no tails); there are three modalities of entry ® membrane fusion (enveloped viruses), direct penetration (injection viral genome), endocytosis (naked viruses, entire virus enters). Inside the cell the capsid is removed = uncoating. - dsDNA: similar to replication human DNA, each strand is used as template - ssDNA: production complementary DNA molecule and then like dsDNA - +ssRNA: used directly as mRNA - -ssRNA: production complementary strand = +ssRNA ® used as mRNA - dsRNA: +ssRNA and -ssRNA separated and then normal replication 5 Enveloped viruses cause persistent infections, naked viruses are released by exocytosis or lysis (slow release). Latency: some viruses remain dormant in cells (ex herpes) ® latent virus/provirus, some viruses are integrated in chromosomes, this integration is permanent. Viruses can cause mutations leading to tumors: carry oncogenes; activate human oncogenes; inhibit human tumor suppressor genes. Ex lymphoma ® human herpes virus 4 Culturing viruses: you can grow bacteriophages in a plate ® formation of viral plaque = dead bacteria, it allows the estimation of their number. Viroids = circular pieces of RNA, cause of disease in plants Prions = endogenous protein that is usually made by alpha helices (PrP proteins); if there’s the introduction of a prion with beta sheets ® causes also alpha helices to become beta, there’s misfolding ® precipitation ® neurodegenerative diseases. Infections Symbiosis = 2 interdependent organisms Mutualism = both organisms benefit Commensalism = only one organism benefits without harming the other Parasitism = one organism benefits and harms the other Normal microbiota can become opportunistic if: introduced in an unusual site in the body; immune suppression; changes in the normal microbiota. Reservoirs of infection: - animal reservoirs: zoonoses (we don’t transmit diseases to animals), very difficult to eradicate - human carriers: we release microbes in the environment during the active disease; asymptomatic infected individuals are called infective carriers. Healthy carriers don’t develop the symptoms - Non-living reservoirs: soil, water and food The exposure to microbes can result in: - Contamination: presence microbes in or on our body - Infection: when the microbes overcome our defenses and invade our body Iatrogenic diseases = caused by medical treatments or procedures / nosocomial diseases = acquired in health care setting. Virulence factors: - Infectivity = ability of microbes to invade and replicate in host’s tissues - Pathogenicity = ability to cause disease - Virulence = ease of infection and degree of pathogenicity - Virulence factors = traits that enable the pathogen to enter the host, adhere to host cells, gain access to nutrients and escape detection by immune system 1. Adhesion factors: allow adhesion to hosts and establishment of colonies, they’re called adhesins in bacteria (target for antibiotics, they can be modified during infection) and attachment proteins in viruses. 2. Extracellular enzymes: help the pathogen to maintain the infection, invade and avoid body defenses ® hyaluronidase (= degrades hyaluronic acid), collagenase (= degrades collagen fibers), coagulase (= activates clotting proteins, microbe hides inside clot), kinase (= digest blood clots releasing microbe and allowing tissue invasion). 3. Toxins: exotoxin are released by microbes in the environment (cytotoxins, neurotoxins and enterotoxins), endotoxins = lipid A 4. Antiphagocytic factors: capsule is made by hyaluronic acid which is not recognized by immune cells as foreign; they also produce chemicals that prevent the fusion of the phagosome with the lysosome 6 Stages of infectious disease: - Incubation period: between infection and appearance of signs and symptoms - Prodromal period: mild symptoms - Illness: most severe signs and symptoms - Decline: signs and symptoms subside, if they don’t ® death - Convalescence: tissue repair Transmission: - Contact transmission: direct ® person to person; indirect ® through fomites (objects); droplet transmission (travel up to 1m) - Vehicle transmission: airborne transmission (aerosols travel for more than 1m); waterborne transmission ® fecal-oral through contaminated water; foodborne transmission ® fecal-oral through contaminated food; bodily fluid transmission ® contaminated blood, urine and saliva - Vector transmission: biological vectors transmit pathogens and serve as host; mechanical vectors are carriers (arthropods) ® the passively transmit pathogens Epidemiology: - Endemic infection: stable incidence in a given area - Sporadic: scattered cases in a given area - Epidemic: greater frequency in a given area - Pandemic: greater frequency in more than one continent Pathogenic gram+ Virulence factors: - Glycocalyx: favors attachment to surfaces forming biofilms, protects from desiccation and protects from recognition - Antiphagocytic factors - Capsule: composed by hyaluronic acid ® doesn’t activate immune response; slippery components prevents phagocytosis - Antiphagocytic chemicals: prevent fusion phagosome with lysosome, ex M protein (+ destabilizes complement) - Enzymes: hyaluronidase, collagenase, coagulase, kinase - Toxins: the host produces antitoxins = neutralizing antibodies; toxic shock syndrome toxin, exfoliative toxin (Staphylococcus) and enterotoxin are superantigens ® activate T cells in an aspecific manner causing a massive release of cytokines (inflammation) - Protein A: binds IgG and inhibits opsonization and the complement cascade - Beta-lactamase: degrade penicillin - C5a peptidase: cleaves C5a ® reduction of chemotaxis - Secretory IgA protease ® destroys secretory IgA (one of the main mechanisms of protection eliminated) Pathogenic gramVirulence factors: - Lipid A: endotoxin, activates TLR4 ® increased expression of inflammatory cytokines ® vasodilation, shock, DIC (septic shock is when both the pre-inflammatory and the antiinflammatory responses are activated) - Iron binding compounds: compete with heme proteins for iron (growth factor for bacteria) - Hemolysins: release nutrients and iron from RBC - Type III secretion system: inserts in the host cell’s membrane and introduces virulence factors into the cell - Neuraminidase (P. aeruginosa): increases the attachment of fimbriae to cell receptors 7
