Microbiology — Bacteria, Viruses and Infection

Bacteria are prokaryotic microorganisms — single-celled, no membrane-bound nucleus, typically 1–10 μm in size. They were the first forms of life on Earth (~3.5 billion years ago) and remain the most abundant organisms on the planet. Cell structure: Cell wall: provides shape and protection. Most bacteria have peptidoglycan cell walls. Critical target for antibiotics (β-lactams, vancomycin). Gram staining: developed by Hans Christian Gram (1884). Gram-positive bacteria have thick peptidoglycan walls that retain crystal violet stain (purple). Gram-negative bacteria have thin peptidoglycan plus an outer lipopolysaccharide (LPS) membrane — lose crystal violet, stain red with safranin. LPS (endotoxin) triggers septic shock. Cell membrane: phospholipid bilayer, site of respiration in bacteria. Capsule: polysaccharide outer layer in some bacteria. Inhibits phagocytosis — major virulence factor. Streptococcus pneumoniae, Klebsiella, Haemophilus influenzae. Flagella: protein filaments for motility. Used to swim toward nutrients (chemotaxis). Pili (fimbriae): protein filaments for attachment to surfaces and host cells. Sex pili transfer plasmid DNA (conjugation). Plasmids: small circular DNA molecules separate from the chromosome. Often carry antibiotic resistance genes. Shapes: Coccus (spherical): Staphylococcus, Streptococcus, Neisseria Bacillus (rod): E. coli, Salmonella, Clostridium, Mycobacterium Spirochete (spiral): Treponema pallidum (syphilis), Borrelia burgdorferi (Lyme disease)

Bacteria reproduce asexually by binary fission — one cell divides into two identical daughter cells. Under ideal conditions, E. coli divides every 20 minutes — from one cell, a billion can be generated in 10 hours. Bacterial genetics: Bacterial DNA is a single, circular chromosome in the nucleoid region (no nuclear membrane). Most genes are constitutively expressed but many are regulated (operons). Horizontal gene transfer (HGT): Bacteria can share genes between cells and even between species — a key mechanism for spreading antibiotic resistance. Transformation: uptake of naked DNA from the environment. Griffith's experiment (1928) demonstrated transformation and led to the discovery that DNA is the genetic material. Transduction: bacteriophage (virus that infects bacteria) accidentally packages bacterial DNA and transfers it to a new bacterium. Conjugation: direct cell-to-cell contact via sex pili. Plasmid DNA transferred. The major mechanism of antibiotic resistance spread — resistance genes on plasmids can spread rapidly between different bacterial species. Bacterial spores: Endospores: formed by Clostridium (C. difficile, C. tetani, C. perfringens, C. botulinum) and Bacillus species under adverse conditions. Metabolically dormant, extraordinarily resistant to heat, drying, disinfectants, and radiation. Can remain viable for centuries. Not killed by standard boiling — require autoclave (121°C, 15 psi). Major infection control challenge. Biofilms: Communities of bacteria embedded in a self-produced matrix on surfaces. Highly resistant to antibiotics and host defences. Form on medical devices (catheters, prosthetic joints, heart valves). Dental plaque is a biofilm. Responsible for the majority of chronic bacterial infections.

Viruses are obligate intracellular parasites — not cells. They cannot replicate independently and are not considered alive by most definitions. Size: 20–300 nm (much smaller than bacteria). Visualised by electron microscopy. Structure: Nucleic acid: DNA or RNA (not both), single or double stranded, linear or circular. The genome. Capsid: protein shell surrounding the nucleic acid. Composed of protein subunits (capsomeres). Symmetrical — icosahedral (e.g., adenovirus), helical (e.g., influenza nucleocapsid), or complex (e.g., bacteriophage T4). Envelope: lipid bilayer derived from the host cell membrane. Present in some viruses (HIV, influenza, herpes, SARS-CoV-2). Envelope disrupted by detergents and alcohol — explains why hand sanitiser is effective against enveloped viruses. Glycoproteins: surface proteins projecting from envelope. Responsible for binding to host cell receptors. HIV gp120 binds CD4. SARS-CoV-2 spike protein binds ACE2. Target of most vaccines and antiviral drugs. Viral replication cycle: 1. Attachment: viral surface proteins bind specific host cell receptors (determines tropism — which cells can be infected) 2. Entry: fusion with plasma membrane (enveloped viruses) or endocytosis 3. Uncoating: viral genome released into cytoplasm 4. Replication: using host ribosomes and energy, new genomes and proteins are synthesised 5. Assembly: new virions packaged 6. Release: by lysis (kills cell) or budding (enveloped viruses acquire envelope while leaving) Viral tropism: HIV infects CD4+ T cells, macrophages, dendritic cells (express CD4 and CCR5/CXCR4 co-receptors). Hepatitis B infects hepatocytes. Rhinovirus infects upper respiratory epithelium. Tropism is determined by receptor expression on host cells.

Pathogenesis is the mechanism by which a microorganism causes disease. Virulence is the degree of pathogenicity. Steps in infection: Exposure → Adherence → Colonisation → Invasion → Immune evasion → Tissue damage → Disease Virulence factors: Toxins: - Exotoxins: proteins secreted by bacteria. Highly potent. Botulinum toxin (most toxic substance known — prevents ACh release), tetanospasmin (prevents GABA/glycine release → spastic paralysis), cholera toxin (activates adenylyl cyclase → massive secretory diarrhoea), Shiga toxin (inhibits ribosome function → haemolytic uraemic syndrome). - Endotoxin (LPS): Gram-negative outer membrane component. Released when bacteria die. Triggers massive cytokine release → fever, vasodilation, septic shock. Adhesins: surface proteins for attachment to host cells. Invasins: proteins that allow bacteria to invade normally non-phagocytic cells. Immune evasion: capsules (inhibit phagocytosis), IgA proteases, complement evasion, intracellular survival (M. tuberculosis survives inside macrophages). Koch's Postulates (1884): Criteria for establishing a microorganism as the cause of a disease: 1. The microorganism must be found in all diseased individuals but not in healthy ones. 2. The microorganism must be isolated from the diseased individual and grown in pure culture. 3. The cultured microorganism must cause disease when introduced into a healthy host. 4. The microorganism must be re-isolated from the experimentally diseased host and shown to be identical to the original. Limitations: some organisms can't be cultured (e.g., T. pallidum), some cause disease only in combination with other factors, molecular Koch's postulates now applied using genetic evidence.

Antimicrobials exploit differences between microbial and human cells. Antibiotic mechanisms of action: Cell wall synthesis inhibitors: Beta-lactams (penicillins, cephalosporins, carbapenems): inhibit transpeptidase (penicillin-binding proteins), preventing peptidoglycan cross-linking. Bactericidal. Selective toxicity: human cells have no cell walls. Vancomycin: binds D-Ala-D-Ala terminus of peptidoglycan precursors. Used for MRSA. Nephrotoxic and ototoxic. Protein synthesis inhibitors: Aminoglycosides (gentamicin): bind 30S ribosomal subunit. Bactericidal. Nephrotoxic and ototoxic. Tetracyclines: bind 30S subunit, block aminoacyl-tRNA attachment. Bacteriostatic. Macrolides (azithromycin, erythromycin): bind 50S subunit. Bacteriostatic. Chloramphenicol: bind 50S subunit. Serious side effect: aplastic anaemia. DNA/RNA synthesis inhibitors: Quinolones (ciprofloxacin): inhibit DNA gyrase and topoisomerase IV. Bactericidal. Rifampicin: inhibits bacterial RNA polymerase. Used for TB. Metronidazole: forms toxic radicals that damage DNA. Used for anaerobes and protozoa. Cell membrane disruption: Polymyxins (colistin): disrupt outer membrane of Gram-negatives. Last resort for MDR organisms. Mechanisms of antibiotic resistance: Enzymatic inactivation: beta-lactamase enzymes (including ESBL and carbapenemases) destroy beta-lactams. The most common resistance mechanism. Target modification: altered PBPs (MRSA), altered ribosomal binding sites (macrolide resistance). Efflux pumps: actively pump antibiotic out of the cell. Reduced permeability: loss of porin proteins in Gram-negatives reduces antibiotic entry. Antiviral drugs: Target viral-specific steps to avoid host toxicity: Nucleoside analogues (aciclovir, tenofovir, remdesivir): mimic nucleosides, incorporated into viral DNA/RNA, terminate chain extension. Protease inhibitors (ritonavir): prevent viral protein maturation. Neuraminidase inhibitors (oseltamivir/Tamiflu): prevent influenza virus release from host cells. Reverse transcriptase inhibitors: target HIV's RT, which lacks human equivalent. Combination antiretroviral therapy (HAART): 3+ drugs targeting different steps prevent resistance. HIV viral load becomes undetectable — patients live near-normal lifespan and cannot transmit the virus (U=U: Undetectable = Untransmittable).