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Antibiotics resistance Conjugation: Antibiotics resistance genes are on the plasmids and the plasmids can be transferred between bacteria via conjugation Transformation: sometimes genes are present in the environment and the bacteria can take it up from the environment, this is called Environmental...

Antibiotics resistance Conjugation: Antibiotics resistance genes are on the plasmids and the plasmids can be transferred between bacteria via conjugation Transformation: sometimes genes are present in the environment and the bacteria can take it up from the environment, this is called Environmental Uptake The third mechanism is transduction where its done by bacteriophages horizontal gene transfer in bacteria, and it involves bacteriophages Infection: A bacteriophage infects a bacterial cell by injecting its DNA into the host cell. Transduction:The bacteriophage's DNA takes over the bacterial cell's machinery, causing the bacteria to produce new phage particles. Accidental Packaging: Sometimes, bacterial DNA fragments are mistakenly packaged into new phage particles along with or instead of the phage DNA. Transfer: When these new phages infect another bacterial cell, they inject the bacterial DNA they carry, leading to the transfer of genetic material from the first bacterium to the second. Antimicrobial susceptibility typing: the two main methods for determining if bacteria are antibiotic resistant are: 1. Disk Diffusion (Kirby-Bauer Test): Antibiotic-impregnated disks are placed on an agar plate inoculated with bacteria. The effectiveness of the antibiotic is indicated by the size of the inhibition zone around the disk. certain sized zones of inhibition allows us to identify the type of bacteria and wither its antibiotic resistant or not  2. Broth Dilution (Minimum Inhibitory Concentration or MIC): This method involves exposing bacteria to different concentrations of an antibiotic in a liquid medium. The lowest concentration that prevents visible bacterial growth is the MIC, indicating the effectiveness of the antibiotic. History of microbiology Antonie van Leeuwenhoek , made first microscope Louis Pasteur Germ theory of fermentation Disproved spontaneous generation – theory of abiogenesis. Organisms like maggots, mice, frogs and agents of food spoilage spontaneously generated from ‘life force’ present in inanimate materials. Robert Koch Developed lab methods for growing microbes Founded laboratory microbiology: agar media, Petri plates, nutrient solutions, aseptic techniques, elucidation of bacterial species and staining techniques. Established link between SINGLE microbe & SPECIFIC disease (Koch’s postulates). Fermentation Pathways Some microbes, known as anaerobes, grow in the absence of oxygen through a process called fermentation. Fermentation is carried out by anaerobic bacteria and involves different pathways. For example, sugars are converted into lactic acid, acetic acid, and propionic acid.  Lactic Acid: Used in the dairy industry, particularly in the production of products like yogurt and cheese.  Yeast (Saccharomyces cerevisiae): Plays a key role in baking and brewing by fermenting sugars to produce carbon dioxide and alcohol. Pasteurisation avoiding the spoilage of milk and wine and also to stop people getting sick from drinking these Difference between pasteurisation and sterilisation Is a proses where milk gets heated up to 70C and the cold down and then stored this proses reduces the number of bacteria, but it doesn’t kill all of them UHD milk has a longer shelf life because its sterilised. KOCH'S POSTULATES 1. The microbe must be present in every case of the disease (especially in 'lesions'). 2. The microbe must be isolated from an individual 'case' and grown in axenic culture. 3. The disease must be reproduced when this pure culture is inoculated into healthy susceptible host. 4. The same microbe should be able to be re‐isolated from the infected host and grown in laboratory culture. Summery;  Presence: Find the microbe in all cases of the disease.  Isolation: Grow the microbe in a pure culture from an infected individual.  Reproduction: Introduce the microbe to a healthy host and reproduce the disease.  Re-isolation: Isolate the same microbe from the newly infected host. Characteristics of living things? Movement (bacteria are with their flagellum but not all bacteria are motile ) Reproduction (they can also reproduce via conjugation, transduction and transformation) Sensitivity (like chemotaxis) Growth Respiration (bacteria can be anaerobic Aswell) Excretion Nutrition Subdivision of cellular organism Prokaryotes Genetic material (DNA) in cytoplasm Bacteria are different from all eukaryotes (animals, plants, fungi, ‘protists’, etc.) Includes organisms always regarded as bacteria –e.g. pathogens Also includes photosynthetic bacteria – e.g. the CYANOBACTERIA (‘blue‐green algae’) Prokaryotic domains – Eubacteria (= Bacteria) – Archaebacteria (= Archaea) – key difference : membrane structure – usually biological membranes are phospholipid bilayer – contain fatty acids – Archaea have complex lipids, isoprene‐based can be linked together to form a monolayer Bacteria and archaea membrane Archaea membrane Classification based on Genetic information When we isolate bacteria, we identify them based on morphological differences and biochemical characteristics. Before the advent of genetic methods, these were the primary techniques used to classify bacteria into different species. This approach was prevalent until the 1990s. In the 1990s, a new technique called multi-locus enzyme electrophoresis (MLEE) was introduced. MLEE analyzes the electrophoretic mobility of multiple enzymes encoded by different loci, providing a more precise method for distinguishing bacterial species and understanding their genetic relationships. Review: Features of bacteria PROKARYOTES: no nucleus. Organisation: typically, unicellular. Diverse metabolism:  Heterotrophs/photoautotrophs aerobes/anaerobes. Cell Size: typically, ~1-2 mm. Internal structure: no membrane-bound organelles. In Eukaryotic cells: organelles are often surrounded by a membrane similar in structure to the cell membrane but with a different composition of protein and phospholipid. Classifying Bacteria Description: prokaryotes, absorbers (what do they use as nutrients) , wet conditions(grow in water or not), animal decomposers, cell walls, unicellular Types: eubacteria, Archaebacteria, Gram‐negative, Gram‐positive, acid fast(they contain mycolic acid which makes the cell was more though allowing it to survive more environmental stress ), cyanobacteria Morphology: cocci, bacilli, spirals, etc. Nutrient Type: chemoheterotrophs, photoheterotrophs, chemoautotrophs, photoautotrophs Durable state: endospores (some) Diseases: tetanus, botulism, gonorrhea, Chlamydia, tuberculosis, etc., etc., etc. Ecological requirements: Oxygen, temperature, gender , since 1990s we sequenced 7 housekeeping genes allowed to understand evolution via multi locus enzyme electrophoresis , in 2005 on ward a new technique was developed called hole genome sequencing or next generation sequencing and by 2012 It become common to sequence the entire genome of bacterise Energy Capturing Metabolism of Microorganisms: Microorganisms are categorized based on their energy-capturing metabolism into two main groups: autotrophs and heterotrophs. 1. Autotrophs: o Autotrophs use inorganic compounds to convert them into organic compounds. There are two subtypes of autotrophs:  Photoautotrophs: These include green sulfur bacteria, purple sulfur bacteria, cyanobacteria, and algae. They use light as a source of energy.   Chemoautotrophs: These use inorganic compounds and do not necessarily rely on light energy for their metabolic processes. 2. Heterotrophs: o Heterotrophs rely on organic compounds for their energy needs. There are two subgroups of heterotrophs:  Photoheterotrophs: These organisms need organic compounds and generally use organic molecules for energy but sometimes can use sunlight as a supplementary source.  Chemoheterotrophs: These organisms exclusively use organic compounds for their energy requirements. Cell Shape (aka Morphology) - limited range  two most common: 1) Spherical Cells cocci (pl.), coccus (sing.) may aggregate: – chains - ‘streptococci’ – clumps - ‘staphylococci’ – Pairs - diplococci Neisseriae Spherical Cells: Division planes and cell arrangements 2) Rods bacilli (plural), bacillus (sing.) – from Latin - ‘stick’ – hence Bacillus genus e.g. Bacillus anthracis But… many other rods exist - so use ROD (not bacillus) Structures of the bacterial cell Capsule or Mucilage layer  Also called Glycocalyx  Not essential for cell viability  Found in some bacteria  Outside cell wall  When well define -> capsule  When less defined ->Slime layer  Bactera that use capsule grow in small colonies  Usually, a single polysaccharide   Polysaccharide type can help identify species or strain  Functions of capsule/mucilage 1. ADHERENCE (capsule is sticky) - ‘biofilms’ 2. PROTECTION against water loss 3. PROTECTION against phagocytes (white cells) in pathogenic bacteria 4. PROTECTION against chemicals e.g. disinfectants 5. They help bacteria to adhere to different surfaces the pathogens form biofilm which helps to form capsules which are sticky the capules protect bacteria from water and macrophage Bacterial Cell Wall Structure: major component PEPTIDOGLYCAN or murein Long (s)GLYCAN chains with repeating sub-units of: (i) N-acetylglucosamine(G) and (ii) N-acetylmuramic acids (M)  cross-linked by short PEPTIDE chains Underneath the capsule is the bacterial cell wall, which is essential for bacteria. The cell wall is primarily composed of a major component called peptidoglycan (or murein). The peptidoglycan is made up of two repeating units: N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). The peptidoglycan monomers are synthesized in the cytosol of the bacterium where they attach to a membrane carrier molecule called bactoprenol. The bactoprenols transport the peptidoglycan monomers across the cytoplasmic membrane and work with other enzymes to insert the monomers into existing peptidoglycan enabling bacterial growth after the binary fission. The M and G subunits alternate and the peptide chains are connected by lysine interpeptide cross bridges PEPTIDOGLYCAN UNIQUE polymer (unique polysaccharide & peptide components) forms a single molecule surrounding cell  very strong, yet permeable because they are need netruattds transported and secretion of proteins like toxins target for anti-bacteria attack: penicillin lysozyme Two major types of cell wall due to major differences in cell wall structure (electron microscopy) two groups termed ……………… GRAM-POSITIVE and GRAM-NEGATIVE The bacterial envelope (wall + membranes) Gram-positive bacteria relatively thick cell wall (>20 nm) high internal osmotic pressure (turgid) high peptidoglycan content (>50%) wall contains other polymers (teichoic acids) typically sensitive to lysozyme and penicillin no further layers outside the cell wall Surface polymers such as teichoic acids play important roles in cell shape determination, regulation of cell division, pathogenesis and antibiotic resistance. The bacterial envelope (wall + membranes): Gram-negative bacteria relatively thin cell wall (100 metres in soil) Branched - 2D or 3D growth Chitin also found in the exoskeleton of crustaceans and insects. The chemical structure show the glucose structure with an attached amine group. The monomers will bind together by a glycosidic linkage between the glucose molecule  Not composed of ‘standard cells’ unlike yeast Multinucleate compartments, Coenocytic form [“common cell”] – where cytokinesis doesn’t occur despite multiple nuclear division Cross-walls they aren’t true cross wall [SEPTA] have pores, the pores allow nuclei to apical through this allows apical growth happen it also mean nutrients can be move between them Cytokinesis is the separation of replicated cells. In hyphae this doesn’t occur fully and instead it forms septa. These septa have gaps in them and this allows movement between the compartments. The pores allow the nuclie to pass through it so it allowes this apical tip growth Hyphae aggregate [visible]:  on surface of substrate (mould = growing mycelium)  ‘fruiting bodies’ (mushrooms & toadstools)  An alternative to hyphe where they form fruite and boody   Hyphae spread out in 3 dimensions from the base resulting in visible 3 dimensional fungi Mycelium on food Mycelium: The rhizoid mycelium and the fruiting body are aggregates of hyphae that form the visible surface of the fungus. The mycelium is the main vegetative part of the fungus and is responsible for growth, nutrient absorption, and reproduction.  1) MOULDS - filamentous fungi: 1. Aerobes: o Definition: Moulds are mostly aerobes, meaning they require oxygen for their respiratory metabolism. 2. Decomposers: o Role: They are major decomposers of biopolymers in soil and water. o Function: They break down organic matter, recycling nutrients back into the ecosystem. 3. Harmless vs. Pathogenic: o Mostly Harmless (Necrotrophs):  Necrotrophs: These fungi grow on dead organic matter, playing a key role in decomposition. o Few Pathogens (Biotrophs):  Biotrophs: These fungi grow on living matter and can be considered pathogenic as they derive nutrients from living hosts. YEASTS – non filamentous Unicellular fungi Grow by (i) budding or (ii) fission So if they grow by buddying they form a lot of individual ballistoconidia If they grow by fission, you can have it all connected to each other there is a higher chance of a pseudo hyphae Schizosaccharomyces - a fission yeast Schizosaccharomyces is a fission yeast so it forms via fission a type of replication where the cell grows and replicates by lengthening. This then forms a new cell wall by cytokinesis which allows the separation of the two daughter cells Yeast  Most organelles in animal cells also found in yeast cells  e.g. nucleus, ribosomes (80S), mitochondria, endoplasmic reticulum, Golgi body, etc.  Cell wall is major additional feature  COLONIES on agar media  Slower growth than many bacteria (doubling time 2-3 h)  Colonies take 1-3 days to form  Doubling time is in comparison to bacteria which typically have a doubling time in the frame of 20-30 minutes Antifungal Agents and Their Mechanisms: 1. Polyenes: o Mechanism: Polyene antibiotics work by targeting and binding to ergosterol, a key component of fungal cell membranes. This binding disrupts the cell membrane's integrity, leading to increased permeability and ultimately, cell death.  o Examples:  Amphotericin B: A systemic antifungal used for severe infections.  Nystatin: Used topically due to its toxicity if ingested; it's effective for treating localized fungal infections. 2. Azole Antifungals : o Mechanism: Azole antifungals inhibit the enzyme lanosterol 14-α-demethylase, which is essential for the synthesis of ergosterol. This inhibition depletes ergosterol in the cell membrane, disrupting membrane structure and function. o Common Types: Azole antifungals are widely used due to their broad-spectrum activity and relatively lower toxicity compared to polyenes.  Examples:  Fluconazole: Used for treating systemic and superficial fungal infections.  Itraconazole: Effective against a variety of fungal pathogens.  Clotrimazole: Commonly used topically for skin and mucous membrane infections. Azole class – Inhibit fungal ergosterol biosynthesis Mechanism of Action of Fluconazole and Azoles: 1. Ergosterol Depletion: 1. Depletion of ergosterol affects the fluidity of the fungal cell membrane. Ergosterol is analogous to cholesterol in human cell membranes and is crucial for maintaining membrane integrity. 2. Fluconazole: 1. Fluconazole inhibits cytochrome P450, an enzyme required for the action of lanosterol 14α-demethylase. This enzyme is responsible for the demethylation of lanosterol to form ergosterol. 3. Azole Inhibitors: 1. Azoles, including fluconazole, inhibit the active site of lanosterol 14α-demethylase, blocking the demethylation of lanosterol to ergosterol. This inhibition leads to reduced membrane fluidity and the accumulation of toxic sterol intermediates. 4. Lanosterol Demethylation: 1. In normal cells, lanosterol demethylase removes the methyl group from lanosterol, forming ergosterol. Ergosterol is vital for the fungal cell membrane’s structure and function. 5. Mechanism of Inhibition: 1. Fluconazole and other azoles bind to the active site of lanosterol 14α-demethylase, reducing or stopping the production of ergosterol. Without ergosterol, the cell membrane loses its fluidity and structural integrity. 6. Cell Membrane Breakdown: 1. The lack of ergosterol causes the fungal cell membrane to break down due to turgid pressure, leading to cell lysis and death. The build-up of toxic sterol intermediates further contributes to the antifungal effect. Ergosterol Biosynthesis and the Mechanism of Azole Antifungals: The top section of the diagram illustrates the normal ergosterol biosynthesis pathway. Squalene, a precursor to steroids, is converted to lanosterol. The enzyme lanosterol 14-α-demethylase then converts lanosterol to ergosterol. Ergosterol is a crucial component of the fungal cell membrane, maintaining its fluidity and integrity. When azole antifungals are introduced, they enter the fungal cell via passive diffusion (this mechanism is proposed). Once inside, they inhibit the demethylase enzyme, preventing the conversion of lanosterol to ergosterol. As a result, lanosterol accumulates within the cell. This accumulation is detrimental because sterols, when not properly converted, are toxic to the cell membrane. The presence of excess lanosterol alters the membrane's permeability, disrupting cellular functions and ultimately leading to the termination of fungal growth. Polyene class - Amphotericin B Can also be used to treat protozoal infections Mechanism of action is when the amphotericin binds to the cell membrane bound ergesterol. When eight of these associated modules gather it is proposed that they form an ion channel within the cell membrane causing efflux of ions from within the cells rendering the cell useless and ultimately dies. Due to the similarity between ergesterol (found in fungal membranes) and cholesterol (found in mammalian membranes) amphotericin B can affect host cells also leading to side effects. However ergesterol has a high affinity for amphotericin that cholesterol so it should bind more to the fungius than the host cells.  DIMORPHIC FUNGI  Both cellular forms meaning they can be harmfull or yeast  Two structural phases: 1 yeast phase (unicellular) or 2 filamentous phase (hyphae) Depending on the condition and they can be disgused as yeast then turn filamentuse Candida albicans:(kan-di-da-al-bi-kans) This dimorphism is often what causes the pathogenicity of these type of fungi. Most dimorphic fungi are commensal, that is they are found in nature, including on the body, as yeasts. They are controlled by the immune system and never grow beyond what the body can tolerate. When they turn filamentous they become invasive pathogens and can spread very quickly. In candida the yeast to hyphae switch is caused by mild temperature change or exposure to antibiotics as a protective mechanism against the drug. Yeast form is thought to disseminate quicker in the blood and hyphae to invade tissues. REPRODUCTION BY SPORES  May be ASEXUAL or SEXUAL  Spore structure:  (1) nucleus  (2) dehydrated cytoplasm, (3) glycogen & (4) thick spore wall  They contaminate everything  Usually coloured - survive in sunlight (air dispersal) they are resisitant to heat , dryness and sunlight Asexual spores – two main types Conidia and Conidiophores: 1. Individual Spore - Conidium (pl. Conidia): o Definition: Conidia are asexual spores produced by certain fungi. o Location: Found on aerial hyphae known as conidiophores. o Release: Conidia are released into the air. o Diagnostic Use: They are diagnostic for particular fungi, helping to identify them. 2. Conidiophore: o Definition: Specialized aerial hyphae that bear conidia. o Reproduction Site: Reproduction occurs at the terminal end of conidiophores, leaving them as stalks after conidia are released. o Desiccation and Survival: Conidia can survive desiccation and sunlight, allowing them to spread through the air. Sporangiospores and Sporangiophores: 1. Multiple Spores in a Sac - Sporangiospores: o Definition: Sporangiospores are asexual spores produced in a sac called a sporangium. o Location: Found on aerial hyphae known as sporangiophores. o Release: Sporangiospores are released into the air when the sporangial wall splits or through the complete release of the sporangium. 2. Sporangiophore: o Definition: Specialized aerial hyphae that bear sporangia. o Reproduction Site: Reproduction occurs within the sporangium, which eventually splits or releases the sporangiospores. Differences Between Conidia and Sporangiospores:  Conidia: Individual spores produced on conidiophores, released directly into the air.  Sporangiospores: Multiple spores produced in a sporangium, released through the splitting of the sporangial wall or the release of the entire sporangium. Asexual reproduction: Asexual Reproduction: Imperfect Fungi: Characteristics: Only produce asexual spores. Perfect Fungi: Characteristics: Capable of both asexual and sexual reproduction. Sexual Spores: There are many different types of sexual spores. Sexual Reproduction: Three Main Groups: Zygomycetes: Sexual Reproduction: Involves the fusion of hyphae from two individuals to form a zygospore. Zygospore: Develops a thick, resistant wall. Example: Mucor (commonly known as bread mould).  Environmental Role: Often environmental contaminants (e.g., bread mould) but can cause zygomycosis in rare situations. Reproduction in Rhizopus: Fusion: Two closely positioned hyphae with haploid chromosomes fuse to create a diploid cell. Meiosis: These diploid cells undergo meiosis, resulting in two haploid cells. Metabolically Inert: These haploid cells remain inactive until conditions become favourable for growth.  Ascomycetes (Sac Fungi): Also Known As: Sac fungi Sexual Spores: Produced in a sac-like structure called an ascus. Number of Spores: Each ascus contains eight ascospores. Release Mechanism: The ascus bursts open to release the ascospores into the air. Basidiomycetes: Sexual Spores: Produced on a specialized cell called a basidium.  Number of Spores: Each basidium produces four basidiospores. Release Mechanism: Basidiospores are released into the air upon maturation. Basidiomycetes:  The most complex fruiting bodies are seen in basidiomycetes  (mushrooms and toadstools)  Products (Mycotoxins) 1. Ergot (Claviceps purpurea in cereals):  Description: Alkaloid toxin related to lysergic acid diethylamide.  Occurrence: Most often seen in rye but can be found in other grasses such as wheat.  Transformation: An alkaloid toxin is transformed in the liver as part of detoxification, producing pyrrole structures that alkylate nucleic acids and proteins, thereby changing their structure and function. 2. Aflatoxins (Aspergillus flavus) in Stored Foods:  Effects: Causes liver damage, necrosis, cirrhosis, and carcinoma.  Metabolism: Aflatoxins are metabolized in the liver to reactive epoxide groups, which are carcinogenic and mutagenic. 3. Amanita phalloides - ‘Death Cap Fungus’:  Type: A basidiomycete.  Toxins Produced: Amatoxins and phallotoxins.  Action: Inhibit RNA polymerase, halting protein synthesis in the liver. 4. Psilocybin – Magic Mushrooms:  Psychoactive Component: Psilocybin.  Conversion: Biologically inert psilocybin is converted to psilocin by dephosphorylation in the liver.  Similarity to Serotonin: Psilocin is similar to serotonin, making it of interest as a psychiatric drug. 5. Antibiotics:  Penicillin and Cephalosporin: o Action: Disrupt the synthesis of peptidoglycan found in the cell wall of bacteria.