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Prokaryotic organisation: no nucleus (a circular chromosome in the nucleoid), 70S ribosomes (30S and 50S subunits, targets of many antibiotics), cytoplasmic membrane without sterols. Cell wall: peptidoglycan (chains of N-acetylglucosamine and N-acetylmuramic acid cross-linked by peptides). Gram-positive: thick peptidoglycan with teichoic acids, stains purple. Gram-negative: thin peptidoglycan plus an outer membrane with lipopolysaccharide (LPS, the endotoxin) and porins, stains pink; the periplasmic space contains beta-lactamases. Mycobacteria have a waxy mycolic acid layer (acid-fast staining). Appendages: flagella (motility), pili or fimbriae (adhesion; sex pili for conjugation), capsule (polysaccharide; resists phagocytosis), and biofilm matrix. Endospores (Bacillus, Clostridium) are dormant, heat-resistant forms.

Binary fission doubles the population each generation time (20 minutes for E. coli in ideal conditions). Growth curve phases: lag, exponential (log), stationary and death. Requirements: carbon and energy sources (autotrophs and heterotrophs), nitrogen, minerals, growth factors, temperature (mesophiles grow at 37 degrees Celsius), pH and oxygen (obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles).

The chromosome is a single circular DNA; plasmids are extra-chromosomal DNA that often carry antibiotic resistance and virulence genes. Horizontal gene transfer: transformation (uptake of naked DNA), transduction (transfer by bacteriophages) and conjugation (plasmid transfer through a pilus). Transposons and integrons move genes between replicons. Bacteriophages are viruses of bacteria: lytic cycle (replication and lysis) or lysogenic cycle (integration as prophage). Phages can transfer toxin genes (diphtheria, cholera and botulinum toxins are phage encoded in some species).

1. Compare the wall of Gram-positive and Gram-negative bacteria. 2. What is LPS? 3. Describe the phases of the bacterial growth curve. 4. Name the three mechanisms of horizontal gene transfer. 5. What is a plasmid?

1. Gram-positive: thick peptidoglycan and teichoic acids. Gram-negative: thin peptidoglycan and an outer membrane with LPS and porins. 2. Lipopolysaccharide, the endotoxin of the Gram-negative outer membrane. 3. Lag, exponential, stationary, death. 4. Transformation, transduction, conjugation. 5. A small circular extrachromosomal DNA, often carrying resistance or virulence genes.

The microbiota is the community of microorganisms living on and in the body (skin, mouth, gut, vagina); the gut has the most (mainly Bacteroidetes and Firmicutes). Functions: colonisation resistance against pathogens, digestion of fibre and production of short-chain fatty acids and vitamins K and B, training of the immune system. Dysbiosis (imbalance) is associated with Clostridioides difficile infection after antibiotics, inflammatory bowel disease, obesity and allergy. Probiotics are live beneficial microorganisms; prebiotics are substrates (inulin, fibre) that favour them; faecal microbiota transplantation treats recurrent C. difficile infection.

Targets: cell wall synthesis (beta-lactams, vancomycin), protein synthesis at the 30S (aminoglycosides, tetracyclines) or 50S (macrolides, chloramphenicol, linezolid) subunit, nucleic acid synthesis (quinolones on DNA gyrase, rifampicin on RNA polymerase), folate synthesis (sulfonamides, trimethoprim) and membrane function (polymyxins, daptomycin). Resistance mechanisms: enzymatic inactivation (beta-lactamases, aminoglycoside-modifying enzymes), target modification (PBP2a in MRSA, altered ribosome or gyrase), reduced uptake (porin loss) and efflux pumps. Resistance genes spread on plasmids. Antibiotic stewardship limits overuse.

Steps of infection: transmission (contact, droplet, airborne, faecal-oral, vector, vertical), adhesion (pili, adhesins), colonisation and invasion, evasion of host defences (capsule, protein A, antigenic variation, intracellular survival), and damage. Toxins: exotoxins are secreted proteins with specific actions (diphtheria toxin blocks protein synthesis; tetanus and botulinum toxins act on neurotransmitter release; cholera toxin raises cAMP; superantigens of Staphylococcus aureus and Streptococcus pyogenes cause toxic shock). Endotoxin (LPS) causes fever, and septic shock in large amounts. Biofilms are communities of bacteria in a matrix on surfaces (catheters, prostheses) that resist antibiotics and immune defences.

1. Give two functions of the gut microbiota. 2. Which antibiotics target the 30S and 50S subunits? 3. Explain how MRSA resists beta-lactams. 4. Difference between exotoxin and endotoxin? 5. Why are biofilms clinically important?

1. Colonisation resistance and production of short-chain fatty acids (also vitamin synthesis and immune training). 2. 30S: aminoglycosides and tetracyclines. 50S: macrolides, chloramphenicol, linezolid. 3. It produces PBP2a, an altered penicillin-binding protein with low affinity for beta-lactams. 4. Exotoxins are secreted proteins with specific actions; endotoxin is LPS from the Gram-negative outer membrane and causes non-specific effects such as fever and shock. 5. They protect bacteria from antibiotics and immunity, causing persistent device-related infections.

Staphylococcus aureus (catalase positive, coagulase positive; clusters): skin infections (boils, impetigo), wound infections, endocarditis, osteomyelitis, toxin diseases (food poisoning, toxic shock syndrome, scalded skin); MRSA is a major hospital pathogen. S. epidermidis (coagulase negative) infects catheters and prostheses through biofilms. Streptococci (catalase negative; chains): classified by haemolysis and Lancefield groups. Group A (S. pyogenes): pharyngitis, impetigo, scarlet fever, erysipelas, necrotising fasciitis; post-infectious rheumatic fever and glomerulonephritis. Group B (S. agalactiae): neonatal sepsis and meningitis. S. pneumoniae (alpha-haemolytic, capsule): pneumonia, meningitis, otitis; the leading cause of community-acquired pneumonia.

Clostridium tetani (anaerobic; tetanospasmin blocks inhibitory neurotransmitter release causing spastic paralysis; prevented by vaccination), C. botulinum (botulinum toxin blocks acetylcholine release causing flaccid paralysis), C. perfringens (gas gangrene; alpha toxin), C. difficile (antibiotic-associated colitis). Bacillus anthracis (anthrax: cutaneous, inhalational; capsule and toxins). Mycobacteria: M. tuberculosis (slow growing, acid-fast; primary infection in the lung, granuloma with caseous necrosis, latent infection and reactivation; multi-drug therapy) and M. leprae.

Fungi are eukaryotes with chitin walls and ergosterol membranes. Yeasts (Candida, Cryptococcus) and moulds (Aspergillus, dermatophytes) exist, and some are dimorphic. Infections: superficial (dermatophytes: tinea; Candida: thrush, vaginitis), and invasive infections in immunosuppression (invasive candidiasis, aspergillosis, cryptococcal meningitis, Pneumocystis pneumonia in HIV). Diagnosis by microscopy, culture, antigen tests (galactomannan, beta-D-glucan).

Examples: Plasmodium (malaria; Anopheles mosquito vector; falciparum is the deadliest; cyclical fever), Entamoeba histolytica (amoebic dysentery and liver abscess), Giardia lamblia (diarrhoea from contaminated water), Toxoplasma gondii (cats; congenital infection and encephalitis in immunosuppression), Trichomonas vaginalis (sexually transmitted), Trypanosoma and Leishmania (vector-borne).

1. How do S. aureus and S. epidermidis differ? 2. Which toxin causes tetanus and what does it do? 3. What are the features of tuberculosis granulomas? 4. Name two opportunistic fungal infections in AIDS. 5. Which protozoon causes malaria and what is its vector?

1. S. aureus is coagulase positive and more virulent; S. epidermidis is coagulase negative and infects devices via biofilm. 2. Tetanospasmin blocks release of inhibitory neurotransmitters, causing spastic paralysis. 3. Epithelioid macrophages and giant cells with central caseous necrosis. 4. Pneumocystis pneumonia and cryptococcal meningitis (also candidiasis). 5. Plasmodium species; Anopheles mosquito.