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Bacteriology - Full Notes

Source: Medical Microbiology 9e & Jawetz Melnick & Adelberg's Medical Microbiology 28e

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SECTION OUTLINE

1. Bacterial Classification, Structure, and Replication
2. Bacterial Metabolism and Genetics
3. Mechanisms of Bacterial Pathogenesis
4. Role of Bacteria in Disease
5. Laboratory Diagnosis of Bacterial Diseases
6. Antibacterial Agents
7. Gram-Positive Cocci (Staphylococcus, Streptococcus)
8. Gram-Positive Bacilli (Bacillus, Listeria, Corynebacterium, Mycobacterium)
9. Gram-Negative Cocci (Neisseria)
10. Gram-Negative Bacilli (Enterobacteriaceae, Pseudomonas, Haemophilus, Vibrio, Campylobacter)
11. Anaerobes (Clostridium, Bacteroides)
12. Spirochetes, Mycoplasma, Rickettsia, Chlamydia

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1. BACTERIAL CLASSIFICATION, STRUCTURE, AND REPLICATION

Prokaryotes vs. Eukaryotes

Key clinical point: These differences are the basis for selective antimicrobial action. 70S ribosomes are targeted by aminoglycosides, macrolides, tetracyclines, and chloramphenicol. Peptidoglycan is targeted by beta-lactams and vancomycin.

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Bacterial Classification

• Macroscopic/microscopic appearance - colony morphology, Gram stain, acid-fast stain
• Growth/metabolic properties - fermentation patterns, hemolysis, enzyme production
• Antigenicity - O (somatic), H (flagellar), K (capsular) antigens
• Genotype - 16S rRNA sequencing (gold standard for phylogenetics), DNA G+C content

Gram Stain Mechanism

1. Crystal violet (primary stain) - stains all bacteria purple
2. Iodine (mordant) - fixes dye to cell wall
3. Acetone/alcohol (decolorizer) - washes out dye from thin-walled gram-negative cells
4. Safranin (counterstain) - stains gram-negatives pink/red

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Bacterial Cell Wall Structure

Peptidoglycan (Murein)

• Backbone: alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) linked by β-1,4 glycosidic bonds
• Cross-linking: peptide chains from NAM residues, cross-linked by transpeptidase (penicillin-binding proteins, PBPs)
• In S. aureus: pentaglycine bridge connects the peptide chains
• In E. coli: diaminopimelic acid directly links the chains
• Lysozyme cleaves the β-1,4 bond between NAM and NAG (found in tears, saliva, neutrophil granules)
• Beta-lactam antibiotics block transpeptidase (PBP), preventing cross-linking

Gram-Positive Components

• Teichoic acids: phosphate-containing polymers anchored to peptidoglycan (wall teichoic acids) or cytoplasmic membrane (lipoteichoic acids)
  • Act as PAMPs (pathogen-associated molecular patterns) - trigger TLR-2 activation
  • Promote adhesion to host cells
• Protein A (S. aureus): binds Fc region of IgG - prevents opsonization
• M protein (S. pyogenes): antiphagocytic

Gram-Negative Components

• Outer membrane: asymmetric bilayer
  • Inner leaflet: phospholipids
  • Outer leaflet: lipopolysaccharide (LPS = endotoxin)
• LPS structure:
  • Lipid A: the toxic component - activates TLR-4, triggers septic shock
  • Core polysaccharide: inner and outer cores
  • O antigen (O polysaccharide): the outermost, serotype-determining antigen
• Porins: outer membrane proteins forming channels for small molecules (≤700 Da)
• Periplasmic space: between inner and outer membranes; contains enzymes (beta-lactamases), binding proteins
• Gram-negative bacteria are generally more antibiotic-resistant due to outer membrane as a barrier

Bacteria with Alternative Cell Walls

• Mycobacteria: mycolic acids in the cell wall - acid-fast staining (Ziehl-Neelsen: carbol fuchsin, decolorized with acid-alcohol, counterstained with methylene blue)
• Mycoplasma: no cell wall - naturally resistant to beta-lactams; smallest self-replicating organisms
• L-forms: bacteria that have lost cell walls (often under antibiotic pressure)

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External Structures

• Capsule: polysaccharide (exception: Bacillus anthracis - poly-D-glutamate capsule)
  • Antiphagocytic - major virulence factor
  • Detected by Quellung reaction (specific Ab + capsule = swelling)
  • Examples: S. pneumoniae, H. influenzae, N. meningitidis, K. pneumoniae
• Flagella: protein (flagellin) helical propellers
  • Monotrichous (1 polar), Lophotrichous (tuft at one pole), Amphitrichous (both poles), Peritrichous (all around)
  • H antigens used in serotyping (e.g., E. coli O157:H7)
• Pili (Fimbriae):
  • Type I fimbriae: mediate attachment to host cells
  • Sex pili (F pili): mediate conjugation - transfer of plasmid DNA
  • Type IV pili: twitching motility; used by N. gonorrhoeae, P. aeruginosa
• Biofilm: formed when bacteria reach quorum. Protects from antibiotics and host defenses. Important in chronic infections (P. aeruginosa in cystic fibrosis, S. epidermidis on prosthetics).

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Bacterial Replication

• Binary fission
• Generation time: E. coli ~20 min under ideal conditions; M. tuberculosis ~20 hours
• Growth curve phases: Lag → Log (exponential) → Stationary → Death
• Spores: formed by Clostridium spp. and Bacillus spp. under nutrient deprivation
  • Structure: core → cortex (modified peptidoglycan) → spore coat → exosporium
  • Extremely resistant to heat, desiccation, disinfectants, UV radiation
  • Contain dipicolinic acid (stabilizes DNA against heat)
  • Germination triggered by heat shock + specific nutrients

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2. BACTERIAL METABOLISM AND GENETICS

Metabolic Requirements

• Carbon/energy sources:
  • Chemoautotrophs: inorganic compounds as energy
  • Chemoheterotrophs: organic compounds (most pathogens)
  • Phototrophs: light energy
• Oxygen requirements:
  • Obligate aerobe: requires O₂ (M. tuberculosis, P. aeruginosa)
  • Facultative anaerobe: grows with or without O₂ (E. coli, most clinical pathogens)
  • Obligate anaerobe: killed by O₂ (Bacteroides, Clostridium - lack superoxide dismutase and catalase)
  • Microaerophile: requires low O₂ (Campylobacter, H. pylori)
  • Aerotolerant anaerobe: grows without O₂ but not killed by it (Lactobacillus)
• Temperature:
  • Psychrophiles: cold (below 20°C)
  • Mesophiles: 20-45°C (most pathogens, optimum ~37°C)
  • Thermophiles: >45°C

Energy Production

• Glycolysis (Embden-Meyerhof-Parnas pathway): glucose → pyruvate; net 2 ATP
• Fermentation: pyruvate reduced to lactate, ethanol, or other products (no O₂ needed)
  • Mixed acid fermentation (E. coli): produces formate, acetate, lactate, ethanol, H₂, CO₂
  • Butanediol fermentation (Klebsiella, Serratia): positive Voges-Proskauer test
• Aerobic respiration: pyruvate → acetyl-CoA → TCA cycle → oxidative phosphorylation → 38 ATP
• Siderophores: chelate iron from the environment; important virulence factor (e.g., aerobactin in E. coli)

Bacterial Genetics

DNA Transfer Mechanisms

1. Transformation: uptake of naked DNA from environment (Griffith's experiment; competent cells)
2. Transduction: bacteriophage-mediated gene transfer
   • Generalized transduction: any gene transferred (phage accidentally packages host DNA)
   • Specialized transduction: specific genes near phage integration site (e.g., lambda phage)
3. Conjugation: direct cell-to-cell transfer via F (fertility) pilus
   • F+ cell (donor) transfers F plasmid to F− cell (recipient)
   • Hfr (high-frequency recombination) cells: F factor integrated into chromosome
   • R factors (resistance plasmids): carry antibiotic resistance genes; major mechanism of spread

Plasmids

• Extrachromosomal circular ds-DNA
• R (resistance) plasmids, virulence plasmids, bacteriocin plasmids, metabolic plasmids

Mutations and Selection

• Spontaneous mutation rate: ~10⁻⁶ to 10⁻⁹ per base pair per generation
• Antibiotic resistance emerges by selection of spontaneous mutants

Gene Regulation

• Operons: coordinately regulated gene clusters
  • Lac operon (inducible): repressor blocked by allolactose inducer; CAP-cAMP enhances transcription when glucose is absent
  • Trp operon (repressible): repressor activated by tryptophan; also regulated by attenuation
• Two-component regulatory systems: sensor kinase (outside stimulus) → response regulator (inside)
• Quorum sensing: density-dependent gene expression via autoinducers (acyl-homoserine lactones in gram-negatives; peptides in gram-positives)
• Pathogenicity islands: clusters of virulence genes acquired by horizontal gene transfer; often flanked by IS elements; different G+C content from rest of chromosome

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3. MECHANISMS OF BACTERIAL PATHOGENESIS

Virulence Factors

Exotoxins vs. Endotoxin

Important Exotoxins

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4. ROLE OF BACTERIA IN DISEASE (Selected Organisms)

GRAM-POSITIVE COCCI

Staphylococcus

• Toxic Shock Syndrome (TSS): TSST-1 superantigen; tampon-associated; fever, rash, hypotension, multi-organ failure
• Scalded Skin Syndrome: Exfoliatin A/B cleave desmoglein 1 in skin → superficial bullae
• Food poisoning: Heat-stable enterotoxin (A-E); preformed toxin ingested; vomiting ± diarrhea within 1-6 hours

Streptococcus

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GRAM-POSITIVE BACILLI

Bacillus

• B. anthracis: anthrax; non-motile, encapsulated (poly-D-glutamate), spore-forming; anthrax toxin (PA + LF + EF); cutaneous (most common), inhalational ("woolsorters' disease," most lethal), GI
• B. cereus: food poisoning; two toxins: emetic toxin (preformed, heat-stable; rice) and diarrheal toxin (heat-labile, LT-like)

Listeria monocytogenes

• Gram-positive rod; facultative intracellular; characteristic tumbling motility; beta-hemolytic; CAMP test+
• Grows at 4°C (cold enrichment) - contaminated cold foods (deli meats, soft cheeses)
• Listeriolysin O (LLO): pore-forming toxin, allows escape from phagosome into cytoplasm
• ActA: promotes actin polymerization for cell-to-cell spread ("comet tails")
• At-risk: neonates, pregnant women, elderly, immunocompromised
• Treatment: ampicillin (+ gentamicin for severe cases)

Corynebacterium diphtheriae

• Gram-positive, club-shaped rods in "Chinese letter" or "V/L" arrangement
• Metachromatic granules (Babes-Ernst/volutin granules) stain with Albert's stain
• Diphtheria toxin: encoded by corynephage β; ADP-ribosylates EF-2 (elongation factor 2) → stops protein synthesis in all cells
• Pseudomembrane in throat (fibrin, neutrophils, bacteria); if removed, bleeds
• "Bull neck" appearance from cervical lymphadenopathy
• Complications: myocarditis, polyneuropathy
• Treatment: antitoxin + penicillin/erythromycin

Mycobacterium

1. Droplet inhalation → alveolar macrophages phagocytose bacilli
2. Bacilli resist killing: cord factor inhibits phagosome-lysosome fusion; LAM inhibits IFN-γ
3. CD4 T-cells activated → IFN-γ → macrophage activation → granuloma formation
4. Granuloma = epithelioid macrophages + Langhans giant cells + lymphocytes + fibrous capsule
5. Central caseous necrosis (cheese-like) is hallmark
6. Latent TB: granulomas contain bacilli; positive TST/IGRA; no symptoms
7. Reactivation: immunosuppression, malnutrition, HIV → liquefactive necrosis → cavity formation → spread

• ≥5 mm: HIV+, close contact, abnormal CXR
• ≥10 mm: immigrants, healthcare workers, high-risk populations
• ≥15 mm: general population

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GRAM-NEGATIVE COCCI

Neisseria

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GRAM-NEGATIVE BACILLI - ENTEROBACTERIACEAE

• ETEC (Enterotoxigenic): LT + ST toxins; traveler's diarrhea; watery
• EPEC (Enteropathogenic): "attaching and effacing" lesions; infantile diarrhea; no toxin
• EIEC (Enteroinvasive): invades colonic epithelium like Shigella; dysentery
• EHEC (Enterohemorrhagic) O157:H7: Shiga-like toxin 1 and 2; bloody diarrhea → HUS (hemolytic uremic anemia, thrombocytopenia, renal failure); NO ANTIBIOTICS (increase toxin release)
• EAEC: aggregative adherence; persistent diarrhea

• Fecal-oral; 1-2 weeks incubation
• Week 1: bacteremia, "stepladder" fever, rose spots (salmon-colored macules on trunk), relative bradycardia, leukopenia
• Week 2: continued fever, hepatosplenomegaly
• Week 3: complications - intestinal perforation, hemorrhage
• Diagnosis: Blood culture (week 1), stool culture (week 2-3), Widal test (Ab titers, less specific)
• Treatment: Fluoroquinolones (ciprofloxacin) or ceftriaxone; chloramphenicol historically
• Chronic carriers: bile duct (especially with gallstones)

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GRAM-NEGATIVE BACILLI - OTHER

Pseudomonas aeruginosa

• Obligate aerobe; oxidase+; non-fermenting; produces pyocyanin (blue-green pigment); fruity grape-like odor
• Motile with polar flagellum; type III secretion system injects ExoS, ExoT, ExoU into host cells
• Exotoxin A: ADP-ribosylates EF-2 (same mechanism as diphtheria toxin)
• Biofilm (alginate) in CF patients' lungs - chronic infection, impossible to eradicate
• At risk: cystic fibrosis, burns, neutropenia, ICU patients, prolonged hospitalization
• Treatment: anti-pseudomonal beta-lactam (piperacillin-tazobactam, cefepime, meropenem) + aminoglycoside; ciprofloxacin

Haemophilus influenzae

• Small gram-negative coccobacillus; requires Factor X (hemin) and Factor V (NAD) for growth
• Satellite growth around S. aureus colonies on blood agar
• Grows on chocolate agar (heated blood releases X and V factors)
• Type b (Hib): polyribitol phosphate (PRP) capsule; prevented by conjugate vaccine
• Non-typeable strains: most common cause of otitis media, sinusitis, COPD exacerbations
• Hib diseases: meningitis (children <5, peak 6-12 months), epiglottitis ("thumb sign" on X-ray), pneumonia

Vibrio cholerae

• Gram-negative, comma-shaped, polar flagellum; oxidase+; O1 and O139 serogroups cause epidemics
• Cholera toxin (CT): A+B subunit; B binds GM1 ganglioside on intestinal epithelium; A subunit ADP-ribosylates Gsα → constitutive activation of adenylyl cyclase → ↑cAMP → massive Cl⁻ and water secretion
• Rice-water stools: massive watery diarrhea (up to 1 L/hr); no inflammatory cells
• Dehydration is the cause of death; treatment = oral rehydration therapy (ORT)
• Antibiotics (doxycycline) shorten illness

Campylobacter jejuni

• Microaerophilic, curved/S-shaped gram-negative rod; oxidase+; grows at 42°C (selective advantage over normal flora)
• Most common bacterial cause of diarrhea in developed countries
• Source: undercooked poultry
• Bloody diarrhea; self-limited; treat with azithromycin
• Post-infectious complications: Guillain-Barré syndrome (molecular mimicry between LOS and gangliosides GM1)

Helicobacter pylori

• Microaerophilic, curved rod; urease+++ (most important); oxidase+; flagellated
• Urease produces NH₃ → neutralizes gastric acid → creates local alkaline microenvironment
• Colonizes antrum; causes gastritis, peptic ulcer disease
• CagA (cytotoxin-associated gene A): injected via T4SS; promotes inflammation
• VacA: vacuolating cytotoxin; disrupts epithelial integrity
• Associated with gastric adenocarcinoma (MALT lymphoma - can regress with eradication)
• Diagnosis: urea breath test, stool antigen test, biopsy (CLO test, histology, culture)
• Treatment: Triple therapy - PPI + clarithromycin + amoxicillin (or metronidazole) x 14 days

Bordetella pertussis

• Small gram-negative coccobacillus; strict aerobe; Bordet-Gengou agar (blood-glycerol-potato)
• Whooping cough: catarrhal stage (1-2 wks, contagious) → paroxysmal stage (2-4 wks, "whoop") → convalescent stage
• Pertussis toxin (PT): ADP-ribosylates Giα (inhibitory G protein) → ↑cAMP; also causes lymphocytosis
• Filamentous hemagglutinin (FHA): adhesin
• Treatment: azithromycin/erythromycin (macrolides); DTP/Tdap vaccine

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ANAEROBES

Clostridium

• Tetanus: spastic (rigid) paralysis; toxin enters via peripheral nerves, transported to spinal cord, blocks inhibitory neurotransmitters
• Botulism: flaccid paralysis; toxin absorbed from gut or wound, blocks ACh release at NMJ; descending paralysis, diplopia, dysphagia first

Bacteroides fragilis

• Most common anaerobe in normal colon and in clinical infections
• Unique outer membrane polysaccharide capsule; produces beta-lactamase
• Capsular polysaccharide complex (PSA) stimulates abscess formation
• BFT (fragilysin): metalloprotease cleaves E-cadherin → mucosal invasion
• Treatment: metronidazole, beta-lactam/beta-lactamase inhibitor combinations, carbapenems

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SPIROCHETES, MYCOPLASMA, RICKETTSIA, CHLAMYDIA

Treponema pallidum (Syphilis)

• Cannot be cultured in vitro; too thin to visualize by Gram stain; detected by dark-field microscopy
• Primary: painless chancre (hard, indurated) at inoculation site + regional lymphadenopathy
• Secondary: maculopapular rash including palms and soles, condylomata lata, mucous patches, flu-like illness; high infectivity
• Latent: early (<1 yr) and late (>1 yr); asymptomatic
• Tertiary: gummas, cardiovascular syphilis (aortitis → aortic aneurysm), neurosyphilis (tabes dorsalis, Argyll Robertson pupil)
• Congenital: early (snuffles, rash, hepatosplenomegaly) and late (Hutchinson's teeth, interstitial keratitis, saber shin, saddle nose)
• Serology: Nontreponemal (VDRL, RPR) - screening, titer follows treatment; Treponemal (FTA-ABS, TPPA) - confirmatory, remain positive for life
• Treatment: Benzathine penicillin G IM (all stages except neurosyphilis); neurosyphilis: IV penicillin G

Borrelia

• B. burgdorferi (Lyme disease): vector = Ixodes tick; reservoir = white-footed mouse
  • Stage 1: Erythema migrans (target/bull's-eye rash), flu-like
  • Stage 2: cardiac (AV block), neurological (Bell's palsy, meningitis - "aseptic")
  • Stage 3: arthritis (large joints, especially knee), encephalopathy
  • Treatment: doxycycline (early); ceftriaxone IV (late/neuro/cardiac)
• B. recurrentis (Relapsing fever): body louse (Pediculus humanus); antigenic variation of Vmp proteins → episodic fever

Leptospira interrogans

• Spirochete; zoonotic; survives in water contaminated with animal urine (rodents, dogs)
• Weil's disease: severe form - jaundice, renal failure, hemorrhage (conjunctival suffusion)
• Treatment: penicillin or doxycycline

Mycoplasma pneumoniae

• No cell wall → not visible on Gram stain; beta-lactam resistant
• "Walking pneumonia" - atypical pneumonia (community-acquired): gradual onset, dry cough, low-grade fever; CXR worse than clinical picture
• Cold agglutinins (IgM anti-I RBC antigen) - positive in ~50%
• Extrapulmonary: hemolytic anemia, bullous myringitis, erythema multiforme, Steven-Johnson syndrome, encephalitis
• Treatment: macrolides (azithromycin), doxycycline, fluoroquinolones (NO beta-lactams!)

Rickettsia

• Weil-Felix test: cross-reacting antibodies against Proteus OX strains (historical test)
• Treatment: doxycycline (all rickettsial diseases, including in children for RMSF)

Chlamydia

• Elementary body (EB): infectious, metabolically inactive, stable outside cell; infects host cell
• Reticulate body (RB): replicating form, metabolically active, inside inclusion; cannot infect

• Treatment: azithromycin (single dose for urogenital); doxycycline; NOT beta-lactams (no cell wall synthesis)

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5. LABORATORY DIAGNOSIS OF BACTERIAL DISEASES

Microscopy

• Gram stain: most important rapid test
• Acid-fast stain (Ziehl-Neelsen): Mycobacteria, Nocardia (weakly acid-fast)
• Dark-field microscopy: Treponema pallidum, Leptospira
• India ink: Cryptococcus neoformans capsule (not a bacterium, but used in CSF)
• Giemsa stain: intracellular organisms (Rickettsia, Ehrlichia, Chlamydia, Plasmodium)
• Silver stain (Warthin-Starry): T. pallidum, H. pylori, Bartonella
• PAS stain: Tropheryma whipplei (Whipple's disease)

Culture Media

Key Biochemical Tests

Advanced Diagnostics

• PCR: nucleic acid amplification; fastest; detects non-viable organisms; good for slow-growers (TB, Chlamydia)
• MALDI-TOF MS: mass spectrometry for rapid organism identification from colonies
• 16S rRNA sequencing: gold standard for phylogenetic identification
• Serology (ELISA, IFA): antibody detection for Lyme, syphilis, Rickettsia, Bartonella
• Antigen detection: urinary antigen (Legionella, S. pneumoniae), C. diff toxin EIA

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6. ANTIBACTERIAL AGENTS (Overview)

By Mechanism of Action

Cell Wall Synthesis Inhibitors

Protein Synthesis Inhibitors (30S Ribosome)

• Aminoglycosides (Gentamicin, Tobramycin, Amikacin): bind 30S, cause misreading; bactericidal; aerobic gram-negatives; nephrotoxic, ototoxic
• Tetracyclines (Doxycycline, Minocycline): block aminoacyl-tRNA binding to 30S; bacteriostatic; atypicals, Rickettsia, Borrelia; chelate divalent cations - not with dairy/antacids

Protein Synthesis Inhibitors (50S Ribosome)

• Macrolides (Azithromycin, Clarithromycin, Erythromycin): bind 23S rRNA of 50S; block translocation; atypicals, gram-positives; CYP3A4 inhibition (erythromycin/clarithromycin)
• Clindamycin: binds 50S; anaerobes, gram-positives; risk of C. diff colitis
• Chloramphenicol: inhibits peptidyl transferase (50S); bacteriostatic; aplastic anemia risk; "gray baby syndrome" (immature liver UGT)
• Linezolid: oxazolidinone; binds 23S rRNA, prevents 70S initiation complex; MRSA, VRE; MAO inhibitor properties

DNA/RNA Synthesis Inhibitors

• Fluoroquinolones (Ciprofloxacin, Levofloxacin, Moxifloxacin): inhibit DNA gyrase (topoisomerase II) in gram-negatives and topoisomerase IV in gram-positives; bactericidal; do not give with antacids; tendinopathy, QT prolongation; contraindicated in pregnancy/children
• Rifampin: inhibits bacterial RNA polymerase (β subunit); meningococcal prophylaxis, TB (part of RIPE); resistance develops rapidly if used alone; induces CYP450
• Metronidazole: reduced by ferredoxin in anaerobes → reactive intermediate damages DNA; anaerobes, C. diff, H. pylori, protozoa; disulfiram-like reaction with alcohol

Cell Membrane Disruptors

• Polymyxins (Polymyxin B, Colistin): bind LPS, disrupt outer membrane; gram-negative (MDR), especially Acinetobacter, Pseudomonas, KPC; nephrotoxic
• Daptomycin: binds gram-positive cell membrane → depolarization; MRSA, VRE bacteremia/endocarditis; myopathy; inactivated by pulmonary surfactant (not for pneumonia)

Antimetabolites

• Sulfonamides: PABA analogue → inhibit dihydropteroate synthetase → block folate synthesis; bacteriostatic
• Trimethoprim: inhibits dihydrofolate reductase; combined with sulfamethoxazole (TMP-SMX = cotrimoxazole) for synergistic sequential blockade of folate; UTI, PCP prophylaxis, Nocardia

Antibiotic Resistance Mechanisms

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QUICK SUMMARY TABLE: HIGH-YIELD BUGS

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