Infectious disease - Lecture notes All PDF

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Introduction to Bacteriology End of year test  Couple example with mechanism Learning outcomes  Describe the difference between Prokaryotes and Eukaryotes  Describe (and illustrate) the cell wall structure for Gram positive and Gram negative bacteria  Describe the procedure for performing the Gr...

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Introduction to Bacteriology End of year test  Couple example with mechanism Learning outcomes  Describe the difference between Prokaryotes and Eukaryotes  Describe (and illustrate) the cell wall structure for Gram positive and Gram negative bacteria  Describe the procedure for performing the Gram stain technique  Describe the main structure of a bacterial organism Bacterial classification  Not belong in the plant or animal kingdom  Protists classification created o Undifferentiated o Mostly unicellular organisms o Don’t form specialized tissues or organs  Higher protists – Eukaryotic o Protozoa, fungi, most algae  True nucleus, nuclear membrane, multiple chromosomes, mitotic replication o Lower Protists – Prokaryotes  All bacteria  Simpler, lack true nucleus, membrane organelles, undergo binary fission replication  Two names: Genus and species  Further subdivided by: o Morphology o Stains o Oxygen requirements o Spore forming Morphology  Size of bacteria o Usual size  Length 1-5 microns  Width 0.2-2 microns  Two principle forms o Spherical-like: cocci

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Rod-like: bacilli

Methods of study  Light microscopy o Dark field  Slender organisms in wet prep o Phase contrast  Fine detail unstained, living organisms  Electron microscopy o Increase resolving power 200x o Shadow casting and negative staining  Surface appendages o Thin section  Cell surface and internal structures o Freeze etching  Cell wall Bacterial structure  Rigid cell wall – complex structure o Surrounds cytoplasmic membrane o Protects against mechanical damage o Responsible for:  Shape, cell division, antigenic differences o Optional features  Capsule, flagella and pili Cell wall  Contains surface macromolecules o Antigens  Host defence  Serological identification in laboratory  Site of action of some antibiotics o Give rigidity to maintain size and shape o Major component is peptidoglycan  Except mycoplasmas o Gram stain reaction based on cell wall differences  Gram positive bacteria o 2 layers o Thick peptidoglycan layer – heat trapping layer o Has teichoic and lipoteichoic acids  Gram negative bacteria o 3 layers o Thin peptidoglycan layer o Has phospholipids, lipoproteins, lipopolysaccharides (endotoxin)  Fats and sugars sit on top of it

Gram positive:

Gram negative:

Gram stain  Make a bacterial smear  Fix cells to the slide with heat o Attach to slide, kills bacteria, allows stain uptake  Stain with basic dye – crystal violet  Fix stain o Treat with iodine-potassium iodide  Reacts with crystal violet to form precipitate  Decolourise with organic solvent o Wash in alcohol  Counter stain paler dye of different colour o Safranin (red)  Gram positive retain crystal violet  Gram negative are decolourised and stain with Safranin o Alcohol stripped away LPS layer

Cytoplasmic membrane  Lipid layer between two protein layers o Hydrophilic on outside o Hydrophobic in between  Semi-permeable  Involved in cellular processes o Energy production  Electron transport o Nutrient and waste transport (facilitated diffusion) o Gas exchange (passive diffusion) o Endospore production Cytoplasmic contents  Nuclear material o DNA chromosome (haploid)  Inclusion bodies o Infolds of the cytoplasmic membrane o Storage  Ribosomes o Involved in protein synthesis o 30S and 50S units  Targets for some antibiotics  Plasmids o Circular non-chromosomal DNA o Some protein coding o Used to exchange genetic information Capsules  Glycocalyx o Slime layer attached to cell wall o Gelatinous o Made of polysaccharide and polypeptides  Protects against phagocytosis o Neutrophils cannot see those with capsules  Enhances adherence  Biofilms  Require special stains Flagella  Thin, whip like structure  Locomotion in some G+ and G- bacteria  Need special staining techniques  See motility in liquid or semi-solid medium Pili or Fimbriae  Help attach bacteria (stick)  Finer filamentous appendages  Only see with electron microscope  Mostly in G- bacteria

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Two types o Short attachment pili (fimbriae)  Numerous  Adhesion o Long conjugation pili (F- or sex pili)  Few  Plasmid exchange – exchange genetic material

Endospores or Spores  Produced by Bacillus spp. and Clostridium spp.  Resistant, dormant survival form  Endospores are quite resistant to high temperatures (including boiling), most disinfectants, low energy radiation, drying, etc.  The endospores can survive possibly thousands of years until a variety of environmental stimuli trigger germination, allowing outgrowth of a single vegetative bacterium Sporulation 1. DNA replicates 2. Septum develops in cytoplasmic membrane 3. Second layer of cytoplasmic membrane forms around the DNA 4. Peptidoglycan layer forms between membranes – cortx 5. Spore coat forms around cortex 6. Outer membrane of lipid and protein forms – exosporium 7. Bacterium shell degrades

Introductory mycology Learning objectives  Be able to explain the difference between fungi and bacteria and how this impacts on the treatment of fungal disease  Know the 3 morphological classes of veterinary-relevant fungi and at least three species from each class  Learn about methods of culturing fungi Mycology must-knows  Fungi are not plants, animals or bacteria o Have a true nucleus o Cell-wall contains chitin, glucans, other glycoproteins and cellulose o No chlorophyll – obtain energy by breakdown of dead or living organic matter  Antibacterial drugs have no effect on, or worsen fungal disease  Outbreaks of fungal disease are rare o Usually just see sporadic cases  Fungal infections can cause unusual clinical presentations o If it looks weird put fungal on your list of differentials  Fungal disease will often be present in immune-compromised animals o Cannot establish diseases in immune-competent animals so most common in immunecompromised

What is a fungus  A feeding system of colourless tubules o Mycelia (singular: mycelium) are components of the thallus  Hypha = a single branch of my mycelium  Cytoplasmic continuity along hyphal tubes Other fungal structures  A variety of spores can be produced o Under certain environmental conditions  Conidia o Contain spores  Conidiophores o Vertical growing mycelium Kingdoms  Plants = photoautotrophic o Synthesise food directly from inorganic compounds using light energy  Animals = heterotrophic o Unable to produce their own food o Feed by consuming other organisms using an internal digestive chamber  Fungi = heterotrophic o Food digested externally o Produce exo-enzymes o Nutrients absorbed into the fungal cells Kingdom fungi  400 spp. pathogenic for animals and humans  Grow aerobically o Some moulds are obligate aerobes o Yeasts are fermentative and may grow anaerobically o Pathogenic fungi can tolerate 37˚C  Tolerant to o High osmotic pressures o Acidic pH o Antibacterial drugs  Fungi are totally unlike bacteria  May even feed fungi Bacterium  Unicellular prokaryote o No nucleus  Chromosome free within cytoplasm  Extra-chromosomal genetic material  E.g. plasmids (circular double stranded DNA)  No specialised cells Fungus  Eukaryote o True nucleus  Genetic material organised within membrane-bound structure

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o Other specialised membrane bound cell organelles There are unicellular and multicellular fungi More like animals than plants or bacteria

Fungal lifestyles  Saprophytic – thrive on dead, organic matter o Majority of species o Involved in decomposition of organic matter  Abundant in soil, on vegetation and in the water o Some species can cause occasional opportunistic infections in animals  Parasitic o Far fewer of these o Dermatophytes  ringworm  Symbiotic o Obligatory associations with other microorganisms o Non-pathogenic  No veterinary relevance Features of fungi  Capsules o Slime = loose polysaccharide gelatinous sheath deposited around the cell wall o May provide protection against phagocytosis o May possess antigens  Stimulate an immune response in host  Cell wall o Rigid structure  A net of polysaccharide micro fibrils (chitin, glucan, mannan and cellulose)  Plant cell wall predominantly contains cellulose and no chitin  Crosslinked by proteins/glycoproteins o Determines fungal morphology  Different cell wall in different species o Species-specific surface antigens  Sometimes used to identify slow-growing or poorly sporulating fungi  Cytoplasmic membrane o Bilayered membrane containing sterols  Dissimilar to bacterial membranes  Similar to other eukaryotes  Fungi utilise ergosterol and zymosterol  Many mammalian cells utilise cholesterol o Therefore, able to target drugs against fungal sterols Fungal reproduction  Involves a change in mycelial morphology o Differentiation of hyphal structure  Asexual structures and sexual structures  Many fungi have complex life cycles o With asexual and sexual stages  Often separated in time

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Arise in dependence upon conditions  Nutrient depletion  Death or resistance of host

Asexual reproduction  Mitosis of a single parent nucleus  Efficient method of reproduction o Some species rarely produce sexual elements  Clinical disease usually involves the asexual forms of the medically important fungi Mechanisms of asexual reproduction  Sporulation and germination o Diploid non-motile spores formed by cytoplasmic cleavage  Budding of yeast cells  Fragmentation of hyphae Fungal spores  Many fungi produce more than one type of spore  Based on dormancy, they are classified as dispersal and survival spores o Dispersal spores  Diploid conidia (macro- or micro-)  Produced in large numbers – separate completely from parent mycelium  Launched by specialist hyphae (conidiophores)  Germinate readily in available nutrients  Limited capacity for dormant survival o Survival spores  Many not completely spate from mycelium  Tend to be large (e.g. Candida species)  Retain substantial nutrient reserves  Germination only occurs  After slow waning of dormancy  Or in response to a specific stimulus Sexual reproduction  Fusion of 2 hyphal nuclei o Followed by meiotic division  Producing haploid spores (meiospores)  Sexual stages of fungi (teleomorphs vs. anamorphs) are of lesser clinical importance o ID of sexual stages is only possible at specialist laboratories o Sexual stage unknown for some fungi Classification of fungi  Complex classification o Based on hyphae and spore morphologies o Phylogenetic studies have led to extensive reclassification in the last decade Fungi of veterinary importance  Three basic morphological types of veterinary relevance o Yeasts

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Moulds Dimorphic

Yeasts  Unicellular eukaryotes, facultative anaerobes o Occasionally produce hyphae  Virulence factor o Reproduce by budding (blastospores) o If buds remain attached to parent cell = pseudohyphae o Oval or spherical  Species of veterinary importance o Candida albicans o Cryptococcus neoformans o Malassezia pachydermatis  Often commensal organisms in low numbers o Skin o Mucous membranes  Yeast colonies are moist or mucoid on agar o Resemble bacterial colonies  Eukaryotic though Moulds  Wide variety  Strict aerobes  Multicellular filamentous fungi o Produce hyphae  Two states o Vegetative mycelium: on surface of substrate  May produce survival spores o Aerial mycelium: rise above surface  Produce smaller reproductive dispersal spores  Mode of spore formation and spore structure used for classification  Growth is apical o Occurring at the tip of the hyphae  Extension zone  Main centre of metabolic activity  Enables fungus to utilise distant food sources  Species of veterinary importance o Aspergillus spp. o Microsporum spp. o Trichophyton spp.  Mould colonies are fluffy or cottony o Mature colony has three zones  Peripheral extension zone - growing  Fruiting/sporing zone  Central aged zone  Extension of colony occurs until either o Nutrient supply is exhausted

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Or inhibitive metabolic bi-products accumulate  Toxic

Dimorphic fungi  Fungi which can exist in hyphal/filamentous or yeast form o Sporothrix schenckii o Histoplasma spp. o Blastomyces dermatitidis o Coccidioides immitus  Temperature-dependent morphology o 37˚C = yeast o 25˚C = mould Mycotoxicosis  Mycotoxins o Secondary metabolites of toxigenic fungi  Aspergillus spp.  Penicillium spp.  Fusarium spp. o Contaminate crops, pasture or stored feed  Heat stable  Unaffected by feed processing techniques Hypersensitivity reactions  Fungal induction of hypersensitivity – immune-complex-induced tissue injury o Usually related to spores o Rare in animals  Can be associated with chronic pulmonary disease in cattle and horses Fungal culture  Sabouraud’s Dextrose agar (SDA) o pH 5.6  slightly acidic pH helps to inhibit bacterial growth o 2.4% yeast extract/peptones and dextrose for nutrients o Antimicrobials may be included  Chlorhexidine, chloramphenicol to prevent bacterial overgrowth  Cyclohexamide prevents growth of opportunistic fungi (and some pathogens)  Dermatophyte test medium (DTM) o SDA with a pH indicator  Red colour change with dermatophytes  Will turn back too yellow if left in the incubator for too long Growing fungi in laboratory  Culture two plates – aerobic o One at 25˚C and one at 37˚C  Slow growers o Will take a few days (yeasts) up to 1-2 weeks (dermatophytes) – examine 2 times a week

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 DTM colour change in 1-2 weeks Some fungi can grow into the agar Usually asexual forms grow on agar

Staining fungi  Examining exudates or tissue (hair, skin) o First clear samples with 10% potassium hydroxide  1 drop KOH onto slide  Add sample  Pass through Bunsen flame  Place cover slip on preparation  Leave for 5-30 minutes  Add lactophenol cotton blue counterstain o Fungal walls resist alkalis but hair/tissue/mucous are degraded  Lacto-fuchsin(pink)/lactophenol cotton blue o Enhance visibility of fungal hyphae  Indian ink o Negative contrast strain  Stains background to highlight microbes o Highlights thick capsule of Cryptococcus neoformans  Gram stain o Useful for detecting presence of yeasts

Bacteriology replication Learning objectives  Describe the process of binary fission  Describe (and illustrate) the four phases of bacterial growth  Describe the requirements and conditions for bacterial growth and the types of media used  Understand and use the terms used to describe conditions and requirements of bacterial growth  Describe the 4 types of bacterial toxins  Describe the cell wall structure of acid fast bacteria  Explain how acid fast bacteria are stained and how this is different from Grams stain  Describe the unique characteristics of mycoplasmas  Describe the unique characteristics of intracellular bacteria Bacterial replication  Reproduce by binary fission o Cell elongate o Transverse cell membrane formed o New cell wall grows inward o Nuclear material distributed o Cell membrane constricts  Some incomplete cleavage  Chains  Clumps

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Filaments – prolonged delay in cleavage in bacilli

Bacterial growth  Under ideal conditions, grow very rapidly o Growth is an increase in number of cells, not size o Cells can double every twenty minutes o Grown in liquid or semi-solid media  Four main phases of growth o Lag o Log or exponential o Stationary o Death or decline  Lag phase o No appreciable increase o Increased metabolic activity o Adapting to new media  Log/exponential o Cells divide at a constant rate – binary fission o Linear relationship between log of cells and time o May be more sensitive to antimicrobial agents  Stationary phase o Decrease in growth rate  Cell numbers stay constant  Exhaustion of nutrients  Accumulation of toxic products  Death or decline phase o Cells die  Incapable of growth in new medium  May show change in staining characteristics  From G+ to G-ve Bacterial colony  Discrete colony  Starts as a single organism o 12-24 hours – small heap Requirements for growth  Carbon o Most require organic nutrients  Carbohydrates, peptides, lipids  Nitrogen o Vary widely in requirements  Amino acids

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Growth factors o Such as coenzymes – synthesised or in growth medium  Inorganic salts o E.g. phosphate, sulphate, N, S, K, Mg, Fe, Mn, Ca Types of media  Liquid (broth) media or Semi-solid (agar plate) media  Basal media o Boiled meat or vegetable proteins  Mixture of fat, protein, carbohydrate o Add animal serum and other goodies  Special carbohydrates, chemicals  Selective media o Differential growth suppression  Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others  Addition of antimicrobials, antifungals, salts, dyes o Examples of selective media include:  Mannitol salts agar (selects against non-skin flora)  MacConkey agar (selects against gram positives)  Eosin-methylene blue agar (selects against gram-positive)  Phenylethyl alcohol agar (selects against gram-negative)  Differential media o Differential appearance  Allows the growth of more than one microorganism of interest but with morphologically distinguishable colonies o Contains a specific substrate and indicator o Examples of differential media include:  Mannitol salts agar (mannitol fermentation = yellow)  Blood agar (various kinds of haemolysis)  MacConkey agar (lactose fermentation = yellow)  Eosin-methylene blue agar (various kinds of differentiation)  Enrichment media o Enriched media is pre-prepared (or equivalent) media to which additional growth factors have been added  These may be added individually or in complex mixtures o Enriched medium therefore may be chemically defined or chemically undefined, simple or complex  Blood agar (various kinds of haemolysis Physical conditions for growth  Oxygen  Temperature  pH  Moisture  Osmotic pressure  Period of incubation  Light

Oxygen (atmospheric conditions)  Strict aerobes (obligate) o Grow only in presence of air or free oxygen (15-20%). o They obtain their energy through aerobic respiration  Microaerophilic o Require a low concentration of oxygen (2% to 10%) for growth, but higher concentrations are inhibitory. o They obtain their energy through aerobic respiration  Obligate anaerobes o Grow only in the absence of oxygen and are often inhibited or killed by its presence. o They didn’t obtain their energy through anaerobic respiration or fermentation  Aero tolerant anaerobes o Cannot use oxygen to transform energy but can grow in its presence. o They obtain energy only by fermentation and are known as obligate fermenters  Facultative anaerobes o Grow with or without oxygen, but generally better with oxygen. o They obtain their energy through aerobic respiration if oxygen is present, but use fermentation or anaerobic respiration if it is absent. o Most bacteria are facultative anaerobes Temperature  Psychrophiles o Cold-loving bacteria o Their optimum growth temperature is between -5C and 15C o They are usually found in the arctic and Antarctic regions and in stream fed by glaciers  Mesophiles o Grow best at moderate temperatures o Their optimum growth temperature is between 25C and 35C o Most common bacteria are mesophilic and include common soil bacteria and bacteria that live in and on the body  Thermophiles o Heat-loving bacteria o Their optimum growth temperature is between 45C and 70C and are commonly found in hot springs and in compost heaps  Hyperthermophiles o Grow at very high temperatures o Their optimum growth temperature is between 70C and 110C o They are usually members of the Archae and are found growing near hydrothermal vents at great depths in the ocean Heat inactivation  Viability o Heat is used to destroy microorganisms o Effect depends on level of moisture  Moist conditions  Inactivation of cell proteins  Dry conditions  Oxidation and charring

pH  Neutrophiles o Grow best at a pH range of 5 to 8  Acidophiles o Grow best as a pH below 5.5  Allaliphiles o Grow best at a pH above 8.5 Other factors  Moisture o Drying in air can ca...