Microbiology — Bacteria, Archaea, Viruses, Fungi, Microbiome

Microbiology is biology of organisms one cannot see without magnification — roughly cells under 100 μm, plus the viruses that infect them. Microbes constitute the great majority of Earth’s biomass (Bar-On, Phillips, Milo 2018 estimated ~70 Gt C in bacteria + ~7 Gt in archaea vs ~2 Gt in all animals), drive every major biogeochemical cycle, run most modern biotech, and cause most infectious disease. This note covers the three-domain tree, cellular and molecular structure of prokaryotes, viruses across the Baltimore classes, antibiotics and resistance, sterilization and microscopy, the human and environmental microbiome, industrial microbiology, extremophiles, and the medical mycology of pathogenic fungi.

1. The three-domain tree of life

Carl Woese and George Fox (1977 PNAS) sequenced small-subunit ribosomal RNA (16S in prokaryotes, 18S in eukaryotes) across the breadth of cellular life and showed that prokaryotes were not a single group. Two profoundly different lineages — Bacteria and Archaea — joined Eukarya as the three domains. The discovery rested on the fact that the ribosomal RNA gene is universal, slowly evolving, and orthologous, so that distance in rRNA sequence reflects total evolutionary distance.

The Last Universal Common Ancestor (LUCA) predates the bacteria–archaea split, ~3.9–4.1 Gya, and is reconstructed (Weiss et al. 2016 Nature Microbiology) as a chemolithotrophic, anaerobic, hyperthermophilic organism close to modern Wood–Ljungdahl-pathway methanogens at hydrothermal sites. Two-domain (eocyte) topologies (Cox, Embley 2008; Williams, Embley 2015) place eukaryotes inside Archaea as sister to Asgard archaea (Lokiarchaeota, Heimdallarchaeota, Thorarchaeota), strengthening endosymbiotic-host-origin models for the eukaryotic cell.

Key contrasts:

• Bacteria — peptidoglycan cell walls, ester-linked fatty-acyl membrane lipids, 70S ribosomes with bacterial-style ribosomal proteins, formyl-methionine initiator tRNA, sensitive to most clinically used antibiotics targeting the ribosome (chloramphenicol, tetracyclines, macrolides, aminoglycosides) and the cell wall (β-lactams, vancomycin).
• Archaea — pseudopeptidoglycan or S-layer walls (no muramic acid), ether-linked isoprenoid lipids (often as monolayers in hyperthermophiles), 70S ribosomes but with eukaryote-like ribosomal proteins and translation initiation, sensitive to anisomycin and diphtheria-toxin-class inhibitors that target eukaryote-style EF-2 homologues. No known archaeal pathogen of humans.
• Eukarya — see cell-molecular-biology.

2. Bacterial cell

2.1 Structure

Bacterial cells are typically 0.5–5 μm in any dimension. Shape categories:

• Cocci — spheres; Staphylococcus (clusters), Streptococcus (chains), Neisseria (diplococci).
• Bacilli — rods; Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa.
• Spirilla — rigid spirals; Spirillum, Campylobacter, Helicobacter.
• Spirochetes — flexible spirals with axial filaments; Treponema pallidum (syphilis), Borrelia burgdorferi (Lyme), Leptospira.
• Vibrios — comma-shaped; Vibrio cholerae.

The cell envelope distinguishes the two Gram-stain phyla. Hans Christian Gram (1884) developed the differential stain that bears his name:

• Gram-positive — thick peptidoglycan layer (20–80 nm), no outer membrane, often with teichoic and lipoteichoic acids; retains crystal violet–iodine complex after alcohol decolorization, stains purple. Staphylococcus, Streptococcus, Bacillus, Clostridium, Listeria, Enterococcus, Corynebacterium.
• Gram-negative — thin peptidoglycan (~7 nm) between an inner plasma membrane and an outer membrane studded with lipopolysaccharide (LPS) on the outer leaflet; loses crystal violet during alcohol, counterstained pink by safranin. E. coli, Salmonella, Pseudomonas, Neisseria, Helicobacter, Haemophilus.

LPS has three regions: lipid A (the toxic moiety — endotoxin recognized by Toll-like receptor 4 on host immune cells, triggers septic shock), core polysaccharide, and O-antigen (highly variable, basis of Salmonella serotyping). Lipid A activation explains why Gram-negative sepsis is so dangerous.

Mycobacteria (M. tuberculosis, M. leprae, M. avium) are technically Gram-positive but have a waxy mycolic-acid-rich cell wall that resists Gram staining; the acid-fast (Ziehl–Neelsen) stain with carbol fuchsin and acid-alcohol decolorization identifies them.

Endospore stain (Schaeffer–Fulton) with malachite green identifies dormant resistant spores produced by Bacillus (anthrax B. anthracis, cereus food poisoning B. cereus) and Clostridium (botulism C. botulinum, tetanus C. tetani, gas gangrene C. perfringens, C. difficile pseudomembranous colitis). Endospores survive boiling for hours; autoclaving is required to kill them.

Other surface structures:

• Capsule — polysaccharide layer outside the wall (Streptococcus pneumoniae, Klebsiella pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b). Major virulence factor: blocks complement and phagocytosis. Polysaccharide capsules are the antigenic target of pneumococcal and meningococcal conjugate vaccines.
• Flagella — rotary motors driven by H+ or Na+ gradient; chemotaxis tumble-and-run behaviour studied by Berg and Brown (1972) in E. coli.
• Fimbriae / pili — short adhesive (type 1 E. coli, P-pili in UTIs), sex pili for conjugation (F pilus), type IV pili for twitching motility (Neisseria, Pseudomonas) and DNA uptake.

Cytoplasm contains the nucleoid (compacted chromosomal DNA — usually one circular chromosome, sometimes linear in Borrelia and Streptomyces, sometimes multiple in Vibrio cholerae), plasmids (small extrachromosomal circles), 70S ribosomes (50S + 30S subunits with 23S/16S/5S rRNA), inclusion bodies (polyhydroxybutyrate, glycogen, polyphosphate, sulfur granules), and in some species magnetosomes (magnetotactic bacteria Magnetospirillum).

2.2 Growth and physiology

In batch culture bacteria pass through four phases:

1. Lag — metabolic adjustment to medium; no net division.
2. Exponential / log — doubling at a constant rate; population N(t) = N0 · 2^{t/Td}.
3. Stationary — nutrient depletion and waste accumulation balance growth and death.
4. Death / decline — viable count falls; some cells form spores or persisters.

Doubling times range from ~20 min for E. coli on glucose-rich LB at 37 °C, to ~3 h for Mycobacterium smegmatis, to ~24 h for M. tuberculosis, to several days for M. leprae (uncultivable in standard media, grown in armadillo footpads).

Metabolic categories by carbon source and energy source:

• Photoautotrophs — light energy, CO2 carbon; cyanobacteria, purple sulfur bacteria.
• Photoheterotrophs — light energy, organic carbon; purple non-sulfur, Rhodospirillum.
• Chemolithoautotrophs — inorganic chemical energy (H2, H2S, S0, NH4+, NO2−, Fe2+), CO2 carbon; Nitrosomonas, Nitrobacter, Thiobacillus, Beggiatoa, Acidithiobacillus.
• Chemoorganoheterotrophs — organic carbon for both; most clinically important bacteria.

Oxygen relationships:

• Obligate aerobes — M. tuberculosis, Pseudomonas aeruginosa.
• Facultative anaerobes — E. coli, Salmonella, Staphylococcus, Streptococcus; ferment when O2 is unavailable.
• Aerotolerant anaerobes — fermentative but indifferent to O2; Lactobacillus.
• Microaerophiles — require <21% O2; Campylobacter jejuni, Helicobacter pylori (~5% O2).
• Obligate anaerobes — killed by O2; Clostridium, Bacteroides, Fusobacterium, methanogens.

Oxygen toxicity arises from incomplete reduction (superoxide O2−, peroxide H2O2, hydroxyl radical OH·). Aerobes defend via superoxide dismutase (SOD), catalase, peroxidase. Obligate anaerobes lack these and are killed by reactive oxygen species.

Fermentation pathways diversify by end-product: lactic acid (Lactobacillus, Streptococcus), mixed acid (E. coli producing acetate + lactate + formate + ethanol + H2 + CO2), butyric acid (Clostridium), propionic acid (Propionibacterium in Swiss cheese — CO2 makes the holes), ethanol (Zymomonas; yeasts in eukaryotes).

2.3 Biofilms

A biofilm is a structured aggregate of bacteria embedded in self-produced extracellular polymeric substance (EPS) — polysaccharides, eDNA, proteins, lipids — adherent to a surface. The discovery that bacteria mostly do not live as free-swimming planktonic cells but as biofilms (Costerton et al. 1995 Annu Rev Microbiol) reframed many areas of microbiology. Biofilms account for an estimated ~80% of chronic and device-related infections (NIH 2002 estimate): catheter-associated UTIs, ventilator-associated pneumonia, prosthetic joint infections, native valve endocarditis, Pseudomonas in cystic-fibrosis lungs, dental plaque, otitis media. Biofilm cells are 10–1000× less susceptible to antibiotics than planktonic counterparts because of (1) diffusion limitation through EPS, (2) physiological dormancy of deep cells, (3) persister-cell formation, and (4) horizontal gene transfer facilitated by close cell packing.

Quorum sensing is the cell-density-dependent signaling that triggers biofilm formation, virulence factor expression, and bioluminescence. Bonnie Bassler’s work on Vibrio harveyi and V. fischeri luxI/luxR autoinducer systems (1990s–2000s) showed inter- and intra-species communication via diffusible AHL (acyl homoserine lactone) and AI-2 autoinducers.

3. Bacterial diversity — clinical and environmental highlights

3.1 Gram-positive cocci

• Staphylococcus aureus — golden-pigmented (carotenoid staphyloxanthin), catalase+, coagulase+; carrier in nares of ~30% of healthy people; skin/soft-tissue infections, endocarditis, osteomyelitis, toxic shock (TSST-1 superantigen), food poisoning (enterotoxins A–E), scalded skin syndrome (exfoliative toxins). MRSA = methicillin-resistant S. aureus via acquired mecA encoding PBP2a, resistant to all standard β-lactams. VRSA = vancomycin-resistant via vanA from Enterococcus.
• Staphylococcus epidermidis — coagulase-negative; skin commensal; catheter and prosthetic-device infections.
• Streptococcus pyogenes — Group A; pharyngitis (“strep throat”), scarlet fever (erythrogenic toxin), impetigo, erysipelas, necrotizing fasciitis, rheumatic fever (post-streptococcal autoimmune), post-streptococcal glomerulonephritis.
• Streptococcus agalactiae — Group B; neonatal sepsis and meningitis.
• Streptococcus pneumoniae — α-hemolytic, optochin-sensitive, polysaccharide capsule; community-acquired pneumonia, otitis media, sinusitis, meningitis. PCV13 / PCV15 / PCV20 conjugate vaccines (Pfizer, Merck).
• Enterococcus faecalis, E. faecium — gut commensals turned nosocomial pathogens; intrinsic resistance to many antibiotics; VRE vancomycin-resistant via vanA–vanG ligases.

3.2 Gram-positive bacilli

• Bacillus anthracis — anthrax; capsule (poly-D-glutamate) and tripartite toxin (protective antigen + lethal factor + edema factor); cutaneous, pulmonary, gastrointestinal forms; weaponization concern.
• Bacillus cereus — food poisoning (rice, emetic and diarrheal forms).
• Clostridium botulinum — botulinum toxin (BoNT) blocks acetylcholine release at the neuromuscular junction by cleaving SNARE proteins; flaccid paralysis. Therapeutic use Botox.
• Clostridium tetani — tetanospasmin blocks inhibitory glycine/GABA release in spinal cord; spastic paralysis.
• Clostridium perfringens — gas gangrene (myonecrosis); α-toxin (phospholipase C).
• Clostridioides difficile (formerly Clostridium) — pseudomembranous colitis after antibiotic-induced dysbiosis; toxin A (enterotoxin), toxin B (cytotoxin); FMT cure rates >85%.
• Listeria monocytogenes — intracellular pathogen; listeriosis from contaminated soft cheese, deli meat, melons; meningitis, stillbirth.
• Corynebacterium diphtheriae — diphtheria toxin (NAD+-dependent ADP-ribosylation of eEF-2) encoded by lysogenic β-phage; airway pseudomembrane.

3.3 Mycobacteria and Actinobacteria

• Mycobacterium tuberculosis — causes ~1.3M deaths/yr (WHO 2023); slow grower (~24 h doubling); Koch identified 1882 (Nobel 1905); latent (tubercle granuloma with caseous necrosis) and active forms; multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains rising. DOTS short-course (HRZE — isoniazid, rifampin, pyrazinamide, ethambutol). Bedaquiline (Sirturo, J&J 2012) and pretomanid (TB Alliance 2019) revived the pipeline.
• Mycobacterium leprae — leprosy / Hansen’s disease; obligate intracellular, prefers cooler skin (~30 °C).
• Streptomyces — soil actinomycetes; source of most clinically used antibiotics (streptomycin, neomycin, tetracycline, erythromycin, vancomycin, rifamycin, daptomycin).

3.4 Gram-negative bacteria

• Escherichia coli — facultative anaerobic gut commensal; pathotypes ETEC (enterotoxigenic, traveler’s diarrhea), EPEC, EHEC / STEC (E. coli O157:H7, Shiga toxin, hemolytic uremic syndrome — the Jack-in-the-Box 1993 outbreak; the German sprout outbreak 2011 O104:H4), EIEC, EAEC, UPEC (uropathogenic, UTI).
• Salmonella enterica — Typhi (typhoid fever), Paratyphi A–C, Typhimurium and Enteritidis (gastroenteritis from poultry, eggs).
• Shigella — bacillary dysentery; type III secretion injection of invasion proteins.
• Campylobacter jejuni — leading cause of bacterial gastroenteritis worldwide; chicken reservoir; Guillain–Barré syndrome (molecular mimicry of ganglioside GM1).
• Vibrio cholerae — cholera; cholera toxin (CT) activates Gs → adenylate cyclase → cAMP → CFTR-mediated Cl− efflux → “rice water” diarrhea; rehydration therapy (ORS, oral rehydration solution) is one of the greatest public-health interventions.
• Vibrio parahaemolyticus, V. vulnificus — shellfish-associated, fatal in cirrhosis patients.
• Helicobacter pylori — gastric ulcer and adenocarcinoma; Barry Marshall and Robin Warren self-experimented (Marshall drank a culture in 1984) and received Nobel 2005.
• Klebsiella pneumoniae — pneumonia and UTI; carbapenem-resistant Enterobacteriaceae (CRE) hyperendemic.
• Pseudomonas aeruginosa — opportunist; cystic fibrosis lung colonization, burn-wound infection, hospital-acquired pneumonia; pyocyanin pigment; intrinsic and acquired multidrug resistance.
• Acinetobacter baumannii — ICU pandrug-resistant strains.
• Neisseria meningitidis — meningococcal meningitis; serogroups A, B, C, W, Y, X covered by various conjugate vaccines.
• Neisseria gonorrhoeae — gonorrhea; antigenic variation of pilus and Opa; widespread cephalosporin resistance now emerging.
• Bordetella pertussis — whooping cough; pertussis toxin (ADP-ribosylates Gi).
• Borrelia burgdorferi — Lyme disease; Ixodes tick vector; identified 1981 Willy Burgdorfer.
• Treponema pallidum — syphilis; cannot be cultured in vitro in standard medium.
• Rickettsia (Rocky Mountain spotted fever, typhus), Chlamydia trachomatis (trachoma, STI), Mycoplasma pneumoniae (walking pneumonia, no cell wall therefore β-lactam-resistant).

4. Bacterial genetics and horizontal gene transfer

Bacteria reproduce by binary fission (clonal), but they routinely exchange DNA horizontally:

• Transformation — uptake of naked environmental DNA by naturally competent cells (Griffith 1928 with S. pneumoniae rough-to-smooth transformation; Avery, MacLeod, McCarty 1944 showed the transforming principle was DNA). Streptococcus, Haemophilus, Neisseria, Bacillus are naturally competent.
• Transduction — phage-mediated transfer. Generalized (lytic phages package random chromosomal DNA, P1 in E. coli, P22 in Salmonella) or specialized (lysogenic phages excise imprecisely, λ in E. coli).
• Conjugation — direct cell-to-cell transfer via sex pilus (F plasmid in E. coli; Lederberg and Tatum 1946, Nobel 1958). Hfr strains integrate F into the chromosome and transfer chromosomal genes.

Mobile genetic elements (plasmids, transposons, integrons, ICEs) ferry antibiotic-resistance and virulence genes across species and across domains.

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats with CRISPR-associated nucleases) is the bacterial and archaeal adaptive immune system against phages and plasmids. Discovered as repeats by Ishino (1987) and Mojica (1993, 2005); identified as immune by Barrangou and Horvath (Danisco, 2007 Science) in Streptococcus thermophilus. Engineered as a programmable nuclease by Jennifer Doudna and Emmanuelle Charpentier (2012 Science), Nobel 2020. See genetics-and-genomics for engineering uses.

5. Antibiotics

5.1 Classes by target and mechanism

Cell wall (peptidoglycan) synthesis

• β-lactams — penicillins, cephalosporins, carbapenems, monobactams. Inhibit transpeptidation by acylating the active-site serine of penicillin-binding proteins (PBPs). Alexander Fleming observed Penicillium notatum lysing S. aureus in 1928; Howard Florey, Ernst Chain, Norman Heatley made it a drug in 1940–1945 (Nobel 1945).
  • Penicillins — penicillin G (narrow Gram+), amoxicillin (extended), ampicillin, methicillin (β-lactamase-resistant, withdrawn for nephrotoxicity), oxacillin, nafcillin, piperacillin (anti-Pseudomonas).
  • Cephalosporins — generations by spectrum. 1st: cefazolin, cephalexin. 2nd: cefuroxime, cefoxitin. 3rd: ceftriaxone, ceftazidime, cefotaxime. 4th: cefepime. 5th: ceftaroline (MRSA-active), ceftobiprole.
  • Carbapenems — imipenem/cilastatin (Primaxin), meropenem (Merrem), doripenem, ertapenem. Broadest β-lactam spectrum.
  • Monobactam — aztreonam (Gram-negative only).
• Glycopeptides — vancomycin (Vancocin), teicoplanin, telavancin, dalbavancin, oritavancin; bind D-Ala-D-Ala terminus of pentapeptide and block transglycosylation/transpeptidation. Gram-positive only. vanA resistance substitutes D-Ala-D-Lac (1000× lower affinity).
• Bacitracin — topical only.
• Lipopeptide — daptomycin (Cubicin, 2003); calcium-dependent membrane depolarization in Gram-positive. Not active in lung (surfactant inactivates).

Protein synthesis (30S ribosomal subunit)

• Aminoglycosides — streptomycin (Selman Waksman, Rutgers, 1943; Nobel 1952), gentamicin, tobramycin, amikacin, neomycin. Bactericidal; misread mRNA, block translocation. Concentration-dependent killing, post-antibiotic effect. Nephrotoxic, ototoxic.
• Tetracyclines — tetracycline, doxycycline (Vibramycin), minocycline, tigecycline (broad-spectrum glycylcycline 2005), omadacycline, eravacycline. Bacteriostatic; block aminoacyl-tRNA binding to A site.

Protein synthesis (50S ribosomal subunit)

• Macrolides — erythromycin (1952), clarithromycin (Biaxin), azithromycin (Zithromax). Block peptidyl transferase / nascent peptide exit tunnel.
• Lincosamides — clindamycin.
• Chloramphenicol — broad spectrum; aplastic anemia risk limits clinical use; still used in some low-income settings and for typhoid.
• Oxazolidinones — linezolid (Zyvox 2000), tedizolid (Sivextro 2014); block 70S initiation complex formation. Active against MRSA, VRE.
• Streptogramins — quinupristin/dalfopristin (Synercid).

Nucleic acid synthesis

• Fluoroquinolones — ciprofloxacin (Cipro), levofloxacin (Levaquin), moxifloxacin (Avelox), delafloxacin. Inhibit DNA gyrase (topoisomerase II) and topoisomerase IV. Black-box warnings for tendinopathy, aortic dissection, peripheral neuropathy.
• Rifamycins — rifampin/rifampicin, rifabutin, rifaximin (gut-restricted, used for hepatic encephalopathy and traveler’s diarrhea), rifapentine. Block RpoB subunit of RNA polymerase.
• Metronidazole (Flagyl) — anaerobe-selective; nitro reduction generates radicals damaging DNA.
• Nitrofurantoin — UTI; multiple damage mechanisms.

Folate synthesis

• Sulfonamides — sulfamethoxazole; Gerhard Domagk discovered Prontosil 1932 (Nobel 1939, declined initially under Nazi pressure). Inhibit dihydropteroate synthase.
• Trimethoprim — inhibits dihydrofolate reductase. TMP-SMX combination (Bactrim, Septra) — sequential blockade.

Other / membrane / new modes

• Polymyxins — colistin (polymyxin E), polymyxin B; cationic detergents disrupting Gram-negative outer membrane LPS. Last-resort drugs for carbapenem-resistant Gram-negative. Nephrotoxic. mcr-1 plasmid-borne resistance (Liu et al. 2015 Lancet Infect Dis) spread worldwide.
• Bedaquiline (Sirturo) — diarylquinoline blocking mycobacterial ATP synthase; MDR-TB approved 2012.
• Pretomanid (Dovprela 2019) and delamanid (Deltyba 2014) — nitroimidazoles for MDR/XDR-TB.
• Cefiderocol (Fetroja 2019) — siderophore cephalosporin trojan-horse for carbapenem-resistant Gram-negative.
• Eravacycline, plazomicin, lefamulin (pleuromutilin), omadacycline — recent broad-spectrum entrants.

5.2 Resistance mechanisms

1. Enzymatic inactivation — β-lactamases hydrolyze the β-lactam ring. Classes A, B, C, D (Ambler). Extended-spectrum β-lactamases (ESBLs, e.g. CTX-M, SHV, TEM mutants) hydrolyze 3rd-generation cephalosporins. KPC carbapenemase (Klebsiella pneumoniae carbapenemase, first reported 1996 North Carolina). NDM-1 metallo-β-lactamase (New Delhi, Yong et al. 2009) hydrolyzes nearly all β-lactams except aztreonam; pandemic spread on plasmids. Aminoglycoside-modifying enzymes (AAC, ANT, APH) acetylate, nucleotidylate, phosphorylate; chloramphenicol acetyltransferase (CAT).
2. Target modification — PBP2a in MRSA (encoded by mecA in the SCCmec cassette) has low affinity for all standard β-lactams. vanA in VRE/VRSA produces D-Ala-D-Lac peptidoglycan terminus. Ribosomal methylation (Erm methyltransferase methylates 23S rRNA A2058) confers resistance to macrolides, lincosamides, streptogramin B (MLSB phenotype). DNA gyrase mutations (gyrA, gyrB) cause fluoroquinolone resistance.
3. Efflux pumps — multidrug-resistance (MDR) pumps actively export antibiotic. RND family (AcrAB-TolC in E. coli, MexAB-OprM in Pseudomonas), MFS, SMR, MATE, ABC superfamilies. Often broad-substrate, so a single pump covers many classes.
4. Reduced permeability — porin loss in Gram-negative outer membranes blocks entry of polar antibiotics (β-lactams, fluoroquinolones).
5. Target overproduction — extra DHPS in sulfonamide resistance.
6. Bypass pathways — substitute folate uptake instead of synthesis.

Resistance genes spread on plasmids, transposons, integrons, and ICEs across species barriers — the resistome concept (D’Costa, Wright et al. 2006). Antibiotic stewardship programmes, surveillance (NHSN, EARS-Net, GLASS WHO), and the One Health approach (human + animal + environment) are the response.

Phage therapy — using lytic bacteriophages to treat bacterial infections, pioneered in the Soviet Union (Eliava Institute, Tbilisi) since the 1920s and reintroduced in Western medicine since ~2017 (Strathdee compassionate-use case, Acinetobacter in San Diego; commercial efforts by Adaptive Phage Therapeutics, Locus Biosciences, BiomX).

6. Pathogenicity

6.1 Virulence factors

• Adhesins — pili, surface proteins (FimH in UTI E. coli).
• Invasins — induce host cell uptake (Yersinia Inv, Listeria InlA/InlB, Salmonella SPI-1).
• Capsules — antiphagocytic.
• Endotoxin (LPS lipid A) — Gram-negative, triggers TLR4 → MyD88/TRIF → NF-κB → cytokine storm; sepsis hallmark.
• Exotoxins — secreted proteins, often AB-toxin architecture (A active, B binding).
  • Diphtheria toxin — ADP-ribosylates eEF-2 → translation arrest → cell death.
  • Cholera toxin (CT) — ADP-ribosylates Gαs → constitutive adenylate cyclase activation → high cAMP → CFTR Cl− efflux → diarrhea.
  • Pertussis toxin — ADP-ribosylates Gαi → loss of inhibitory regulation.
  • Botulinum toxin (BoNT A–G) — zinc protease cleaving SNARE proteins (SNAP-25, syntaxin, VAMP) → blocks ACh release → flaccid paralysis.
  • Tetanus toxin (tetanospasmin) — cleaves VAMP in inhibitory interneurons → unopposed motor neurons → spastic paralysis.
  • Shiga toxin — N-glycosidase on 28S rRNA → translation arrest; HUS triad (microangiopathic hemolytic anemia, thrombocytopenia, acute kidney injury).
  • Superantigens — TSST-1, SEA-E, SpeA crosslink MHC II directly to TCR V_β region → polyclonal T-cell activation → cytokine storm.
• Secretion systems — T1SS (hemolysin export), T2SS, T3SS (injection needle, Yersinia/Salmonella/Shigella/Pseudomonas), T4SS (conjugation + effector injection, Helicobacter pylori CagA), T5SS (autotransporter), T6SS (interbacterial warfare via contractile injection apparatus, Vibrio/Pseudomonas).
• Siderophores — iron-scavenging chelators (enterobactin E. coli, mycobactin Mycobacterium); host counters with lipocalin-2.

6.2 Koch’s postulates

Robert Koch (1882, on M. tuberculosis; Nobel 1905) formalized criteria to assign causation:

1. Microbe found in all cases of disease, absent in healthy.
2. Isolated in pure culture.
3. Causes disease when inoculated into a healthy host.
4. Re-isolated from the experimentally infected host.

Modern equivalents include molecular Koch’s postulates (Falkow 1988) requiring that mutation of the candidate virulence gene attenuate disease and complementation restore it.

7. Archaea

Although no archaeal human pathogen is known, archaea dominate many environments and run distinctive metabolisms:

• Methanogens — Methanobacterium, Methanococcus, Methanosarcina; strictly anaerobic; reduce CO2 + 4 H2 → CH4 + 2 H2O or convert acetate or methylamines to methane. In bovine rumens, paddy soils, anaerobic digesters, deep-sea sediments. Roughly half of global atmospheric methane is biogenic — methanogen output.
• Halophiles — Halobacterium salinarum, Haloarcula, Haloferax; salt-saturated brines (Dead Sea, Great Salt Lake, salterns). Halobacterium membrane bears bacteriorhodopsin (Stoeckenius and Oesterhelt, 1971 Nat New Biol) — a light-driven proton pump that motivated subsequent rhodopsin family discoveries and is the structural ancestor of channelrhodopsins used in optogenetics.
• Thermophiles and hyperthermophiles — Pyrococcus furiosus (100 °C), Sulfolobus (75–80 °C, pH 2), Thermococcus, Methanopyrus (122 °C, highest known). Thermus aquaticus (a thermophilic bacterium, not an archaeon — Yellowstone, Brock 1969 J Bacteriol) was the source of Taq DNA polymerase (Saiki et al. 1988), which made PCR practical and earned Kary Mullis the 1993 Nobel.
• Acidophiles — Picrophilus (pH 0). Alkaliphiles — Natronomonas (pH 11).
• Asgard archaea — Lokiarchaeota etc.; close cellular sisters to Eukarya; some have actin-like cytoskeleton and Sec24-like proteins, consistent with archaeal host origin of eukaryotes (Spang et al. 2015 Nature).
• ANME archaea perform anaerobic methane oxidation in consortium with sulfate-reducing bacteria; major methane sink at ocean seeps.

See inorganic-chemistry for sulfur and nitrogen redox chemistry that drives chemolithotrophy.

8. Viruses

8.1 Structure

A virus is a genome (DNA or RNA, ss or ds, linear or circular, segmented or not) packaged in a protein capsid (icosahedral, helical, or complex/asymmetric) sometimes enclosed in a host-derived lipid envelope with embedded viral glycoproteins. Size 20 nm (parvoviruses, ssDNA, 5 kb) to 1 μm+ (pithoviruses; some Mimiviridae and Pandoraviruses ~1 Mb genomes — Abergel, Claverie). Modern definitions place giant viruses near the “fourth domain” boundary, though they are not cellular.

8.2 Baltimore classification

David Baltimore (1971; Nobel 1975 with Howard Temin, Renato Dulbecco) grouped viruses by genome type and pathway to mRNA:

• I — dsDNA (Herpes, Adeno, Pox, Papilloma, Polyoma).
• II — ssDNA (Parvo).
• III — dsRNA (Reo, including rotavirus).
• IV — ssRNA +sense (Picorna, Flavi, Toga, Corona, Calici).
• V — ssRNA −sense (Orthomyxo, Paramyxo, Rhabdo, Filo, Bunya).
• VI — ssRNA-RT (Retroviridae — HIV, HTLV; reverse-transcribe to integrated dsDNA provirus).
• VII — dsDNA-RT (Hepadnaviridae — HBV; pgRNA reverse-transcribed in capsid).

8.3 Lytic vs lysogenic cycles

Lytic phages and viruses replicate then lyse the host, releasing progeny. Lysogenic (temperate) phages can integrate their genome as a prophage and be passed on with the host until induction (UV, DNA damage). Lysogenic conversion carries virulence genes — diphtheria toxin (β-phage in C. diphtheriae), cholera toxin (CTXφ in V. cholerae), Shiga toxin (Stx phages in EHEC), botulinum toxin in some C. botulinum, scarlet-fever toxins in S. pyogenes. Most discovered virulence factors are phage cargo.

8.4 Major virus families (selection)

dsDNA

• Herpesviridae — large enveloped icosahedral genomes 120–230 kb; latency a hallmark. HSV-1 (oral), HSV-2 (genital), VZV (chickenpox / shingles), EBV (mononucleosis, Burkitt lymphoma, nasopharyngeal carcinoma — first oncogenic human virus, Epstein and Barr 1964), CMV (congenital, transplant), HHV-6/7 (roseola), HHV-8 / KSHV (Kaposi sarcoma).
• Adenoviridae — non-enveloped; respiratory, conjunctivitis, gastroenteritis; vectors for ChAdOx1 (AstraZeneca-Oxford COVID-19 vaccine, Sputnik V Ad26+Ad5, Janssen/J&J Ad26.COV2.S, Cansino Convidecia).
• Papillomaviridae — HPV; >200 genotypes; HPV16, 18 (and 31, 33, 45, 52, 58) cause cervical, anogenital, oropharyngeal cancers; E6 binds p53, E7 binds Rb. Gardasil 9 (Merck) covers 9 types. zur Hausen Nobel 2008.
• Polyomaviridae — SV40 (lab), JC (PML in immunosuppressed), BK (renal transplant), Merkel cell polyomavirus (Merkel cell carcinoma).
• Hepadnaviridae — HBV; partially dsDNA, replicates via RNA intermediate (class VII). HBV vaccine recombinant in yeast (Engerix-B, Recombivax HB) — first recombinant subunit vaccine.
• Poxviridae — variola (smallpox; eradicated 1980 WHO, with last natural case 1977 Somalia; Jenner 1796 cowpox vaccination), monkeypox / mpox (2022 outbreak), vaccinia (vaccine strain). Brick-shaped, ~200 kb genome, cytoplasmic replication.

ssDNA

• Parvoviridae — parvovirus B19 (erythema infectiosum / “fifth disease”, aplastic crisis in sickle cell), AAV (adeno-associated virus, replication-defective, gene therapy vector — Luxturna, Zolgensma, Hemgenix).

dsRNA

• Reoviridae — rotavirus (severe childhood diarrhea worldwide, ~200 000 deaths/yr pre-vaccine); RotaTeq (Merck), Rotarix (GSK). Bluetongue virus in livestock.

+ssRNA

• Picornaviridae — small, non-enveloped, naked +RNA translated directly. Poliovirus (Sabin OPV, Salk IPV), rhinovirus (>160 serotypes, common cold), enterovirus 71 (hand-foot-mouth, neurological), coxsackievirus, hepatitis A virus (Havrix vaccine), foot-and-mouth disease virus (FMDV) of livestock.
• Caliciviridae — norovirus (winter vomiting, cruise-ship outbreaks).
• Flaviviridae — enveloped. Hepatitis C virus (Harvey Alter, Michael Houghton, Charles Rice — Nobel 2020; cured by sofosbuvir/velpatasvir Epclusa, glecaprevir/pibrentasvir Mavyret), dengue (4 serotypes; Dengvaxia, Qdenga), Zika (2015–2016 microcephaly outbreak), yellow fever (17D vaccine since 1937, Theiler Nobel 1951), West Nile, Japanese encephalitis, tick-borne encephalitis.
• Togaviridae — chikungunya (alphavirus, arthralgia), equine encephalitis viruses.
• Coronaviridae — large +RNA ~30 kb. Endemic human coronaviruses (229E, NL63, OC43, HKU1, common colds). SARS-CoV (2003, ~10% CFR, contained), MERS-CoV (2012, dromedary camel reservoir, ~35% CFR), SARS-CoV-2 (2019; >7 M reported deaths and millions more by excess-mortality estimates). mRNA vaccines (BNT162b2 Comirnaty Pfizer-BioNTech; mRNA-1273 Spikevax Moderna) deployed at scale within ~1 yr — Katalin Karikó and Drew Weissman Nobel 2023 for nucleoside-modified mRNA enabling these.
• Retroviridae (technically RT class VI) — HIV-1 and HIV-2 (Montagnier, Barré-Sinoussi Nobel 2008; ART regimens NRTI + NNRTI + INSTI; Truvada / Descovy PrEP), HTLV-1 (adult T-cell leukemia).

−ssRNA

• Orthomyxoviridae — Influenza A (segmented, reassortment yields pandemic strains: H1N1 1918 “Spanish flu” ~50M dead, H2N2 1957, H3N2 1968, H1N1 2009 swine flu, H5N1 highly pathogenic avian, H7N9 China 2013), Influenza B (mainly human), Influenza C (mild). HA-NA nomenclature; oseltamivir (Tamiflu), zanamivir (Relenza), baloxavir (Xofluza).
• Paramyxoviridae — measles (most infectious agent known, R0 ~12–18; MMR), mumps, RSV (Beyfortus nirsevimab approved 2023; Arexvy/Abrysvo vaccines 2023), parainfluenza, Nipah (Bangladesh outbreaks, ~70% CFR), Hendra (Australia).
• Rhabdoviridae — rabies (bullet-shaped, neurotropic, essentially 100% CFR once symptomatic; PEP within hours of exposure is life-saving), vesicular stomatitis virus (also vaccine vector — Ervebo Ebola vaccine).
• Filoviridae — Ebola (Zaire, Sudan, Bundibugyo, Tai Forest, Reston species; West Africa outbreak 2014–2016 ~28 000 cases, 11 000 deaths; Ervebo rVSV-ZEBOV vaccine licensed 2019), Marburg.
• Bunyavirales — hantavirus (Sin Nombre, hemorrhagic fever with renal syndrome, hantavirus pulmonary syndrome), Crimean–Congo hemorrhagic fever (CCHF), Rift Valley fever, severe fever with thrombocytopenia syndrome virus (SFTSV).

8.5 Bacteriophages

T4 (large, dsDNA, 169 kb, complex symmetry tail), λ (lysogeny model since Lwoff, Jacob, Monod), M13 (filamentous, ssDNA, basis of phage display — Smith Nobel 2018), Φ29 (ssDNA-binding study system), MS2 (first sequenced genome, Fiers 1976), Φ174 (Sanger first DNA sequencing 1977). CRISPR-Cas anti-phage defense, and the anti-CRISPR proteins (Acr) phages encode in response, form an arms-race system at the heart of microbiology.

9. Sterilization, disinfection, microscopy

9.1 Killing methods

• Moist heat — autoclave — 121 °C, 15 psi (≈103 kPa above atmospheric), 15–30 min; kills spores. D-value = decimal reduction time (time to kill 90%). F0 = equivalent autoclave time at 121 °C. For prion inactivation: extended cycle (134 °C, 18 min) plus alkaline pretreatment.
• Dry heat — 160 °C × 2 h or 170 °C × 1 h; less effective per unit time than moist heat.
• Pasteurization — 63 °C × 30 min (LTLT) or 72 °C × 15 s (HTST flash); reduces vegetative pathogens, not sporicidal.
• Filtration — 0.22 μm filters retain bacteria; 0.45 μm passes Brucella; HEPA 0.3 μm; ULPA 0.12 μm. Viruses pass standard 0.22 μm — tangential-flow ultrafiltration or virus filters needed.
• UV-C — 254 nm peak absorption by nucleic acids; pyrimidine dimers; surface and water only (no penetration).
• Ionizing radiation — gamma (Co-60) or e-beam; medical device sterilization at 25 kGy.
• Ethylene oxide (EtO) — alkylates DNA and proteins; gas sterilant for heat-sensitive medical devices.
• Chemical disinfectants — sodium hypochlorite (bleach 5000 ppm available chlorine for sporicidal use), 70% ethanol or 70% isopropanol (alcohols denature proteins; ineffective on spores), chlorhexidine gluconate (Hibiclens), iodophors (povidone-iodine, Betadine), quaternary ammonium compounds, peracetic acid, glutaraldehyde (Cidex), o-phthalaldehyde (Cidex OPA), hydrogen peroxide vaporization (Bioquell, STERIS).

The Spaulding classification (1968) distinguishes critical (sterilize), semi-critical (high-level disinfect), and noncritical (low-level disinfect) device categories. Z-value = temperature change required to alter D-value by 10×.

9.2 Microscopy

• Bright-field light microscopy — Abbe diffraction limit d ≈ λ/(2 NA); ~200 nm with visible light + oil immersion NA ≈ 1.4.
• Phase contrast — Frits Zernike (Nobel 1953); refractive-index-based contrast of unstained live cells.
• DIC (differential interference contrast / Nomarski) — pseudo-3D shading.
• Fluorescence microscopy — IF (immunofluorescence with labeled antibodies), FISH (fluorescent in situ hybridization with nucleic-acid probes), GFP/RFP transgenes (Shimomura, Chalfie, Tsien Nobel 2008). Super-resolution: STED (Hell), PALM, STORM (Betzig, Moerner) — Nobel 2014.
• Confocal — pinhole rejects out-of-focus light; optical sectioning.
• Electron microscopy — SEM (surface, ~1 nm), TEM (thin section, sub-nm), cryo-EM (vitrified frozen samples; single-particle reconstruction now resolves macromolecules to <2 Å; Frank, Henderson, Dubochet Nobel 2017). Cryo-ET for cellular tomography.

9.3 Culture and detection

Media types — defined (minimal) vs complex (rich, e.g. LB, TSB, BHI); selective (suppresses unwanted organisms — MacConkey for Gram-negative enterics with bile + crystal violet; PEA / CNA selective for Gram-positive); differential (visual distinction — EMB / MacConkey for lactose fermenters; blood agar for hemolysis patterns α partial, β complete, γ none; chocolate agar with heat-lysed RBC for fastidious Haemophilus, Neisseria); Sabouraud dextrose agar for fungi; Löwenstein–Jensen for mycobacteria; Thayer–Martin (chocolate + VCN antibiotics) for Neisseria. CHROMagar chromogenic substrates for rapid Gram-negative ID.

Biochemical ID — API strips, Vitek 2, Phoenix.

MALDI-TOF mass spectrometry — matrix-assisted laser desorption / ionization on whole cells gives a ribosomal-protein-dominated mass spectrum; matched against a library (Bruker Biotyper, bioMérieux VITEK MS) for species identification within minutes at <$1/sample. Standard in clinical micro labs since ~2010.

Molecular detection — Sanger sequencing of 16S rRNA hypervariable regions (V3–V4); qPCR panels (BioFire FilmArray respiratory, GI, BCID panels; GeneXpert MTB/RIF for TB + rifampin resistance, Cepheid). Metagenomic shotgun sequencing (Karius for plasma cell-free microbial DNA, CosmosID, IDbyDNA / Illumina Explify) is moving from research to clinical adoption for unbiased pathogen ID in sepsis and undiagnosed infection.

10. The microbiome

The Human Microbiome Project (HMP, 2007–2012, ~$170M NIH) and MetaHIT (EU, 2008–2013) catalogued bacterial communities at 18 body sites in ~250 healthy adults. The integrative HMP2 (iHMP) followed disease cohorts (IBD, T2D, preterm birth) longitudinally.

Body-site communities:

• Gut — highest density (~10¹¹–10¹² cells per gram colon content); two dominant phyla Bacillota (Firmicutes; Clostridia, Faecalibacterium prausnitzii, Roseburia, Lactobacillus) and Bacteroidota (Bacteroidetes; Bacteroides, Prevotella); plus Actinobacteriota (Bifidobacterium), Pseudomonadota (Proteobacteria, low in health), Verrucomicrobiota (Akkermansia muciniphila). 100–1000× more genes than the human host.
• Oral — site-specific (tongue dorsum, supra- and sub-gingival plaque, saliva, palate). Streptococcus, Veillonella, Neisseria, Haemophilus, Fusobacterium, Porphyromonas gingivalis (periodontitis).
• Skin — Cutibacterium acnes (formerly Propionibacterium), Staphylococcus epidermidis, Corynebacterium, Malassezia (yeast).
• Vaginal — Lactobacillus crispatus, L. iners, L. jensenii, L. gasseri dominate in health, suppressing pathogens by lactic acid production (pH 3.8–4.5). Bacterial vaginosis = shift to anaerobes (Gardnerella vaginalis, Atopobium vaginae, Mobiluncus).
• Lung — long thought sterile; modern sequencing shows low-biomass community dominated by Prevotella, Veillonella, Streptococcus.

Dysbiosis — community shifts associated with disease — has been linked to IBD (Crohn’s, ulcerative colitis), obesity, T2D, NAFLD, colorectal cancer (Fusobacterium nucleatum), atopic dermatitis, allergy, autism (controversial), depression and anxiety via the gut–brain axis (vagal afferents, SCFAs, neurotransmitter precursors). Causality vs correlation remains an active question in most cases.

Fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection has cure rates >85% (van Nood et al. 2013 NEJM), vs ~30% for repeat vancomycin. The FDA approved Rebyota (Ferring, fecal-derived, 2022) and Vowst (Seres SER-109, lyophilized spore-forming bacteria, oral, 2023) — the first standardized live-biotherapeutic products. Trials in IBD, GvHD, ICI checkpoint-inhibitor response, autism, hepatic encephalopathy.

11. Industrial and applied microbiology

11.1 Fermentation foods

• Beer — Saccharomyces cerevisiae (ale, top-fermenting) or S. pastorianus (lager, bottom-fermenting, cold-tolerant hybrid of cerevisiae × eubayanus); barley malt, hops Humulus lupulus, water, yeast.
• Wine — S. cerevisiae strains on grape sugars; malolactic fermentation by Oenococcus oeni.
• Bread — S. cerevisiae leavens dough by CO2; sourdough adds Lactobacillus sanfranciscensis for lactic acid + flavor.
• Yogurt — Streptococcus thermophilus + Lactobacillus delbrueckii subsp. bulgaricus; lactose → lactate, pH drop denatures casein.
• Cheese — starter cultures (lactococci, lactobacilli, propionibacteria for Swiss eye-forming), rennet (chymosin), aging mold cultures (Penicillium roqueforti blue cheese, P. camemberti white-rind).
• Soy fermentation — soy sauce (Aspergillus oryzae, koji), miso, tempeh (Rhizopus oligosporus), natto (Bacillus subtilis var. natto).
• Kimchi, sauerkraut — heterolactic and homolactic LAB sequence.
• Vinegar — Acetobacter aceti oxidizes ethanol to acetic acid.

11.2 Biotechnology

• Recombinant insulin — Eli Lilly Humulin (Genentech-licensed) 1982, the first recombinant pharmaceutical; produced in E. coli as separate A and B chains then renatured (later as proinsulin and cleaved).
• Recombinant hGH — Genentech 1985 E. coli.
• Erythropoietin (EPO) — Amgen Epogen 1989, CHO cells (post-translational sialylation requires mammalian).
• Monoclonal antibodies — most produced in CHO (Chinese hamster ovary) cell lines, some in NS0 or Sp2/0; top global sales include adalimumab (Humira AbbVie, peak >$20B/yr), pembrolizumab (Keytruda Merck), trastuzumab (Herceptin Roche/Genentech), rituximab (Rituxan Roche/Biogen).
• Vaccine production hosts — recombinant HBsAg in yeast (S. cerevisiae); recombinant HPV VLP in yeast (Gardasil) or insect cells (Trichoplusia ni Hi-5 baculovirus for Cervarix); influenza traditionally in embryonated chicken eggs (~150M eggs annually for global supply) or now in MDCK cells (Flucelvax) or insect-cell expressed HA (Flublok); mRNA-LNP encapsulated lipid nanoparticles (Comirnaty, Spikevax).

11.3 Biofuels and bulk chemicals

• Ethanol — corn (US, ~60 B L/yr) or sugarcane (Brazil); S. cerevisiae on hydrolyzed sugars. Cellulosic ethanol from agricultural residue via consolidated bioprocessing (Clostridium thermocellum) remains technologically marginal at scale.
• ABE (acetone–butanol–ethanol) — Clostridium acetobutylicum (Weizmann process, used for cordite in WW1).
• Algal biodiesel — Nannochloropsis, Chlorella; Synthetic Genomics and ExxonMobil collaboration (2009–2017) wound down without commercial output.
• Lactic acid — Lactobacillus for polylactic acid (PLA) bioplastic (NatureWorks).
• Succinic acid, 1,3-propanediol (DuPont Sorona), isoprene (Genencor) — bulk chemicals via engineered E. coli or Saccharomyces.

12. Environmental microbiology

• Nitrogen cycle bacteria — Rhizobium in legume nodules (alfalfa, soy, clover), Bradyrhizobium, Sinorhizobium, Frankia in actinorhizal plants (alder Alnus, Casuarina); nitrogen fixers free-living: Azotobacter, Clostridium, cyanobacteria (Anabaena azollae in rice paddies). Nitrification (Nitrosomonas, Nitrosospira, Nitrosococcus → NO2−; Nitrobacter, Nitrospira → NO3−) — comammox (Nitrospira inopinata, van Kessel 2015) performs full ammonia → nitrate in a single organism. Anammox (anaerobic ammonium oxidation) by Brocadia, Kuenenia couples NH4+ + NO2− → N2; major nitrogen sink in oceans (Strous, Jetten 1999); engineered in wastewater treatment.
• Sulfur cycle — Desulfovibrio, Desulfobacter (sulfate reducers); Beggiatoa, Thiothrix (chemolithotrophic sulfide oxidizers).
• Anaerobic digestion — sequential hydrolysis → acidogenesis → acetogenesis → methanogenesis converts organic waste to biogas (~60% CH4 + ~40% CO2). Municipal sewage, landfill, agricultural digesters.
• Bioremediation — Deinococcus radiodurans (radiation-resistant), Pseudomonas putida for hydrocarbon degradation, mycorrhizal-assisted phytoremediation, BTEX-degrading consortia.

See environmental-engineering for engineered treatment systems and chemical-process-fundamentals for bioreactor design.

13. Extremophiles and astrobiology

• Hyperthermophiles — Methanopyrus kandleri strain 116 grows at 122 °C (Takai 2008). Adaptations: reverse gyrase (positive supercoiling), thermostable proteins with more ion pairs, hyperthermostable monolayer ether-linked lipids (archaeal).
• Psychrophiles — Colwellia, Polaromonas in polar sea ice and cold deep ocean; antifreeze proteins, flexible cold-adapted enzymes.
• Halophiles — Dead Sea, Great Salt Lake; archaeal Halobacterium accumulates K+ to balance Na+ (“salt-in”); bacterial halophiles synthesize compatible solutes (“salt-out”: glycine betaine, ectoine, trehalose).
• Acidophiles — Picrophilus pH 0; cytoplasm pH ~5; proton pumps + impermeable membranes.
• Alkaliphiles — Bacillus pseudofirmus, Natronomonas pharaonis at pH 11; cytoplasm maintained near neutral.
• Piezophiles — Mariana Trench (~110 MPa) Shewanella benthica, Colwellia.
• Radiation-resistant — Deinococcus radiodurans survives 5000 Gy (humans die at ~5 Gy); efficient DNA-repair (homologous recombination, single-strand annealing) reassembling shattered chromosome from hundreds of fragments.
• Polyextremophiles — Sulfolobus acidocaldarius (80 °C, pH 2), Picrophilus (60 °C, pH 0).

Astrobiology uses extremophiles as analogues for life on Mars (subsurface brines, ice), Europa and Enceladus (subglacial oceans with hydrothermal vents, plumes), Titan (hydrocarbon lakes — speculative non-water-based biochemistry). NASA Astrobiology Institute, Europa Clipper (launched 2024), Mars 2020 Perseverance / Mars Sample Return.

14. Fungi — medical and applied

Fungi form a separate kingdom of eukaryotes; chitinous cell walls, β-glucan and mannan polysaccharides, ergosterol membrane sterol (target of azole and polyene antifungals). Two main morphologies: yeast (single-celled, budding or fission) and mold (multicellular, hyphae forming mycelium). Dimorphic species switch with temperature.

14.1 Pathogenic fungi

• Candida albicans, C. glabrata, C. krusei, C. auris (emerging multidrug-resistant, first identified 2009 Japan) — opportunistic; oral thrush, vaginitis, esophagitis, candidemia in central-line patients.
• Cryptococcus neoformans, C. gattii — encapsulated yeast; cryptococcal meningitis in HIV/AIDS.
• Pneumocystis jirovecii — atypical fungus; PJP pneumonia in AIDS (CD4 <200); TMP-SMX prophylaxis.
• Aspergillus fumigatus, A. flavus, A. niger — molds; invasive aspergillosis in immunocompromised; aspergilloma in pre-existing cavities; ABPA. A. flavus produces aflatoxin B1 (hepatocarcinogen contaminating peanuts, corn, tree nuts).
• Dimorphic endemic mycoses — Histoplasma capsulatum (Ohio–Mississippi River valleys, bat/bird guano), Coccidioides immitis/C. posadasii (San Joaquin Valley fever, southwest US), Blastomyces dermatitidis (Great Lakes, Mississippi watershed), Paracoccidioides brasiliensis (Latin America), Talaromyces marneffei (Southeast Asia, HIV-associated).
• Mucormycosis — Rhizopus, Mucor, Lichtheimia; rhinocerebral form in poorly controlled diabetes and immunosuppression; surge during COVID-19 in India.
• Dermatophytes — Trichophyton, Microsporum, Epidermophyton; tinea infections (ringworm, athlete’s foot, jock itch).

14.2 Antifungals

• Polyenes — amphotericin B (binds ergosterol, forms membrane pores; nephrotoxic; liposomal formulations AmBisome reduce toxicity), nystatin (topical).
• Azoles — fluconazole (Diflucan), itraconazole (Sporanox), voriconazole (Vfend), posaconazole (Noxafil), isavuconazole (Cresemba); inhibit lanosterol 14-α-demethylase (CYP51).
• Echinocandins — caspofungin (Cancidas), micafungin (Mycamine), anidulafungin (Eraxis); inhibit β-1,3-glucan synthase.
• Flucytosine — 5-FC; antimetabolite; combination with amphotericin for cryptococcal meningitis.
• Allylamines — terbinafine (Lamisil); inhibits squalene epoxidase; dermatophytes, onychomycosis.
• Ibrexafungerp (Brexafemme 2021) and olorofim (in clinical trials) — new mechanisms (triterpene glucan-synthase inhibitor, dihydroorotate dehydrogenase inhibitor respectively) targeting azole- and echinocandin-resistant strains.

14.3 Applied fungi

• Saccharomyces cerevisiae — model eukaryote (Nobel-decorated genetics: Hartwell, Nurse, Hunt 2001 cell cycle; Schekman, Rothman, Südhof 2013 vesicle trafficking; Ohsumi 2016 autophagy); brewing; recombinant protein expression (HBV vaccine, HPV VLP).
• Pichia pastoris / Komagataella phaffii — methylotrophic yeast for high-yield secreted recombinant protein production.
• Penicillium chrysogenum — penicillin production.
• Aspergillus niger — citric acid (~2 Mt/yr), industrial enzymes.
• Aspergillus oryzae — koji for soy sauce, sake.
• Fusarium venenatum — mycoprotein (Quorn).
• Ganoderma, Lentinula edodes (shiitake), Pleurotus, Agaricus bisporus — edible mushrooms.

14.4 Plant-pathogenic fungi

• Magnaporthe oryzae — rice blast; causes ~10–30% global rice yield loss in epidemic years.
• Puccinia graminis — wheat stem rust; race Ug99 (Uganda 1999) reemerged as a global threat to wheat.
• Fusarium graminearum — wheat scab (Fusarium head blight); deoxynivalenol mycotoxin.
• Phytophthora infestans — technically an oomycete (Stramenopila, not Fungi), but historically grouped here; potato late blight; Irish Potato Famine 1845–1849.
• Cryphonectria parasitica — chestnut blight, wiped out the American chestnut Castanea dentata in the early 20th century.
• Ophiostoma novo-ulmi — Dutch elm disease.

Mycotoxins of agricultural importance: aflatoxins (Aspergillus), fumonisins (Fusarium), ochratoxin A (Aspergillus, Penicillium), patulin (Penicillium, on apples), ergot alkaloids (Claviceps purpurea on rye — historical ergotism epidemics).

15. Cross-cutting modern methods

• 16S rRNA amplicon sequencing — V3–V4 (or V4 only for shorter reads) hypervariable regions on Illumina MiSeq / NextSeq; analysis pipelines QIIME 2, DADA2, mothur. Replaced OTU clustering with ASV exact-sequence inference.
• Full-length 16S — PacBio HiFi or Oxford Nanopore for species-level resolution.
• Shotgun metagenomics — random fragment sequencing; assembly (megahit, metaSPAdes), binning (CONCOCT, MetaBAT, MaxBin), MAG (metagenome-assembled genome) recovery; functional annotation against KEGG, COG, CAZy.
• Metatranscriptomics, metaproteomics, metabolomics — paired multi-omics for community function rather than composition.
• Single-cell genomics of uncultivated microbes — Candidate Phyla Radiation (CPR) bacteria, DPANN archaea — minimalist symbionts and parasites revealed by metagenomics and SAG.
• Pangenomics — core vs accessory genome; the E. coli pan-genome has >40 000 gene families; core only ~3 000. Roary, PIRATE, PPanGGOLiN.

16. Diagnostic microbiology workflow

A clinical microbiology specimen passes through Gram stain, primary culture, identification, susceptibility testing, and result reporting. Time-to-result has compressed enormously over the past decade:

• Conventional culture + biochemicals — 24–72 h for ID + 24 h for susceptibility.
• MALDI-TOF MS from colony — 1–10 minutes for ID once a colony is available.
• MALDI-TOF directly from positive blood cultures — 30–60 minutes via lysis-and-spin protocols.
• Multiplex PCR panels (BioFire BCID2, GenMark ePlex BCID-GP/GN/FP) — 1 h directly from positive blood culture bottle.
• Rapid phenotypic AST (Accelerate PhenoTest, T2 Biosystems) — 4–7 h for MIC.
• Metagenomic NGS (Karius, IDbyDNA Explify) — 24–48 h from blood/CSF/tissue, no growth required, agnostic to pathogen class.

Antimicrobial susceptibility testing (AST) is reported as MIC (minimum inhibitory concentration, μg/mL) with categorical interpretation susceptible (S), susceptible-dose-dependent (SDD), intermediate (I), resistant (R) per CLSI (US) or EUCAST (Europe) breakpoints, which differ slightly. Methods: broth microdilution (gold standard), Kirby–Bauer disk diffusion (zone diameter inversely related to MIC), Etest strip (gradient strip on agar), automated systems (Vitek 2, Phoenix, MicroScan).

Antibiogram — institution-level summary of % susceptible by organism × antibiotic, updated annually; guides empirical therapy.

17. Vaccinology

Modern vaccine platforms span half-a-dozen technology classes:

• Live attenuated — replication-competent, attenuated by passage (MMR — measles, mumps, rubella; varicella; yellow fever 17D; OPV Sabin polio; BCG M. bovis for TB; live-attenuated influenza FluMist; rotavirus RotaTeq, Rotarix). Single dose often sufficient; contraindicated in immunocompromised and pregnant.
• Inactivated whole organism — formalin-killed (IPV Salk polio; hepatitis A Havrix/Vaqta; whole-cell pertussis [largely replaced]; rabies; some flu).
• Subunit / toxoid — purified protein or detoxified toxin (tetanus toxoid, diphtheria toxoid, acellular pertussis [Pa in DTaP, Tdap], HBsAg recombinant in yeast, recombinant flu HA Flublok).
• Conjugate polysaccharide–protein — capsular polysaccharide chemically linked to a carrier protein (CRM197, tetanus toxoid) to recruit T-cell help. Haemophilus influenzae type b (Hib), pneumococcus (PCV13/15/20), meningococcus (Menactra, Menveo, MenQuadfi), typhoid Vi.
• VLP (virus-like particle) — capsid protein assembled without genome (HPV Gardasil/Cervarix, HBV).
• Viral vector — replication-defective adenovirus or vesicular stomatitis virus carrying a target antigen gene (ChAdOx1-S Vaxzevria, Ad26.COV2.S Janssen, rVSV-ZEBOV Ervebo Ebola).
• mRNA-LNP — synthetic mRNA encoding the antigen, encapsulated in lipid nanoparticles (Comirnaty Pfizer–BioNTech, Spikevax Moderna). Modified nucleosides (pseudouridine, 5-methylcytidine) developed by Karikó and Weissman (Nobel 2023) reduce innate immune activation and improve translation.
• DNA vaccines — plasmid encoding antigen; approved for veterinary use; limited human approvals (Inovio).

Adjuvants boost the immune response: aluminum salts (alum, oldest; in DTaP, hepatitis B, HPV), MF59 oil-in-water emulsion (Fluad), AS01 (liposomes + MPL + QS-21; Shingrix, Mosquirix malaria), AS03 (Pandemrix), CpG-1018 (Heplisav-B), Matrix-M (saponin; Novavax Nuvaxovid, Mosquirix).

Herd immunity threshold = 1 − 1/R0. Highly contagious pathogens require high vaccination coverage: measles R0 ≈ 15, threshold ~93%; SARS-CoV-2 ancestral R0 ≈ 2.5–3, threshold 60–67%; Omicron R0 much higher (~10), threshold ~90% but waning + escape complicate the picture. See probability-fundamentals for compartmental epidemic models.

18. Sepsis and the inflammatory response

Sepsis — life-threatening organ dysfunction caused by dysregulated host response to infection (Sepsis-3 definition, Singer et al. 2016 JAMA); SOFA score ≥ 2 from baseline. Septic shock adds persistent hypotension requiring vasopressors and lactate >2 mmol/L despite adequate fluid resuscitation. Mortality 10–40%. Gram-negative LPS activates TLR4–MD2–CD14, Gram-positive lipoteichoic acid and peptidoglycan signal through TLR2; both converge on MyD88 → NF-κB → TNF-α, IL-1, IL-6 cytokine storm → endothelial dysfunction, capillary leak, DIC. Bundles (Surviving Sepsis Campaign) emphasize blood cultures before antibiotics, early broad-spectrum coverage within 1 h, source control, lactate clearance, and crystalloid resuscitation.

19. Microbiology and adjacent fields

Microbiology overlaps with genetics-and-genomics (model organism genetics, CRISPR origins, plasmid biology), cell-molecular-biology (membrane and ribosome biology), immunology-foundations (host–pathogen interactions and vaccine design), ecology-and-evolution (microbial community ecology, horizontal gene transfer, microbial life-history), biochemistry-foundations (enzymology, metabolic pathways), organic-chemistry-foundations (natural products and drug chemistry), biomaterials (biofilm-resistant surfaces, antimicrobial materials), environmental-engineering (water and wastewater treatment), microfluidics (single-cell microbial sorting and droplet screening), and bioinstrumentation (PCR thermocyclers, MALDI-TOF instruments, flow cytometers, optical-density readers).

Adjacent

• cell-molecular-biology — eukaryotic cell biology and the molecular toolkit shared across domains
• genetics-and-genomics — CRISPR origins, plasmid biology, recombinant DNA technology
• immunology-foundations — host defenses, vaccines, antibody therapeutics
• ecology-and-evolution — microbial community ecology and horizontal gene transfer
• biochemistry-foundations — enzymes, metabolic pathways, fermentation chemistry
• environmental-engineering — anaerobic digestion, nutrient removal, drinking-water disinfection
• biomaterials — biofilm-resistant and antimicrobial surfaces
• probability-fundamentals — sequencing statistics, dilution counting, MIC distributions