13.1 Medical Bacteriology: Gram-Positive, Gram-Negative, and Atypical Bacteria

Fundamental Principles of Gram Staining and Cell Wall Architecture

Bacterial classification relies fundamentally on cell wall structure and Gram stain reaction. The Gram stain protocol involves primary staining with crystal violet, iodine mordant application, decolorization with alcohol/acetone, and counterstaining with safranin. Gram-positive bacteria possess a thick multi-layered peptidoglycan cell wall (containing teichoic and lipoteichoic acids) that traps the crystal violet-iodine complex, appearing purple under microscopic evaluation. Conversely, Gram-negative bacteria have a thin peptidoglycan layer surrounded by an outer membrane containing lipopolysaccharide (LPS), which is extracted during alcohol decolorization, allowing counterstaining with safranin to yield a pink/red appearance.

Lipopolysaccharide consists of three components: Lipid A (the toxic moiety responsible for septic shock, activating complement and cytokine release including TNF-alpha, IL-1, and IL-6), core oligosaccharide, and O-antigen. Understanding this architecture is pivotal for predicting antibiotic susceptibility and understanding microbial pathogenesis.

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Gram-Positive Cocci Differentiation and Pathogenesis

Gram-positive cocci represent major causative agents of skin, soft tissue, respiratory, and systemic infections. Initial differentiation begins with the catalase test, which converts hydrogen peroxide into water and oxygen bubbles. Staphylococcus species are catalase-positive, whereas Streptococcus and Enterococcus species are catalase-negative.

Staphylococci

• Staphylococcus aureus: Catalase-positive and coagulase-positive. Virulence factors include Protein A (binds the Fc region of IgG, inhibiting opsonization and phagocytosis), alpha-toxin, and TSST-1 (toxic shock syndrome toxin 1, a superantigen that cross-links the beta-region of T-cell receptors to MHC class II molecules on antigen-presenting cells, causing massive cytokine release). Methicillin-resistant S. aureus (MRSA) carries the mecA gene, encoding an altered penicillin-binding protein (PBP2a) with low affinity for beta-lactams. S. aureus causes abscesses, endocarditis, osteomyelitis, septic arthritis, and scalded skin syndrome.
• Staphylococcus epidermidis: Catalase-positive, coagulase-negative, and novobiocin-sensitive. Highly adept at producing biofilms (extracellular polysaccharide matrix) on prosthetic devices, intravascular catheters, and artificial heart valves.
• Staphylococcus saprophyticus: Catalase-positive, coagulase-negative, and novobiocin-resistant. A leading cause of uncomplicated urinary tract infections (UTIs) in young, sexually active females.

Streptococci

Streptococcal species are categorized by hemolysis patterns on blood agar (alpha = partial green, beta = complete clear, gamma = non-hemolytic) and Lancefield cell wall carbohydrates.

• Streptococcus pyogenes (Group A Strep): Beta-hemolytic, catalase-negative, and bacitracin-sensitive. Key virulence factors include M protein (inhibits phagocytosis and complement activation) and SPE-A (streptococcal pyrogenic exotoxin A, a superantigen). Causes streptococcal pharyngitis, impetigo, erysipelas, and necrotizing fasciitis. Post-infectious immune sequelae include rheumatic fever (type II hypersensitivity following pharyngitis, featuring mitral valve damage, Aschoff bodies, and elevated anti-streptolysin O titers) and post-streptococcal glomerulonephritis (PSGN) (type III immune complex hypersensitivity following pharyngitis or skin infection, presenting with hematuria, periorbital edema, and subepithelial humps on electron microscopy).
• Streptococcus agalactiae (Group B Strep): Beta-hemolytic, catalase-negative, bacitracin-resistant, and CAMP test positive (produces CAMP factor that enlarges the zone of beta-hemolysis formed by S. aureus). Colonizes the female lower gastrointestinal and vaginal tracts; primary cause of neonatal sepsis, pneumonia, and meningitis.
• Streptococcus pneumoniae: Alpha-hemolytic, optochin-sensitive, and bile-soluble. Appears as lancet-shaped Gram-positive diplococci. Encapsulated by a thick polysaccharide capsule (quellung reaction positive). Primary cause of community-acquired pneumonia, otitis media, sinusitis, and adult meningitis (mnemonic: MOPS).

Enterococci

• Enterococcus faecalis & Enterococcus faecium: Group D streptococci that are bile esculin-positive and grow in 6.5% NaCl. Normal enteric flora capable of causing UTIs, biliary tract infections, and subacute bacterial endocarditis. Vancomycin-resistant enterococci (VRE) harbor the vanA gene, which alters the peptidoglycan cell wall precursor target from D-alanyl-D-alanine to D-alanyl-D-lactate.

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Gram-Positive Bacilli

Gram-positive bacilli are divided into spore-forming and non-spore-forming rods.

Spore-Forming Rods

• Bacillus anthracis: Aerobic rod with a unique poly-D-glutamate polypeptide capsule. Anthrax toxin consists of protective antigen, edema factor (a calmodulin-dependent adenylate cyclase that increases cAMP), and lethal factor (a zinc metalloprotease that cleaves MAP kinase kinase). Causes cutaneous anthrax (painless black eschar) and pulmonary anthrax (woolsorter's disease with hemorrhagic mediastinitis and widened mediastinum).
• Bacillus cereus: Aerobic rod causing food poisoning. Produces a preformed heat-stable emetic toxin associated with reheated fried rice, and a heat-labile diarrheal toxin.
• Clostridium tetani: Anaerobic rod producing tetanospasmin, an exotoxin that cleaves SNARE proteins, blocking the release of inhibitory neurotransmitters (GABA and glycine) from Renshaw cells in the spinal cord. Results in spastic paralysis, lockjaw (trismus), risus sardonicus, and opisthotonos.
• Clostridium botulinum: Anaerobic rod producing preformed botulinum toxin, which cleaves SNARE proteins to inhibit acetylcholine (ACh) release at the neuromuscular junction. Causes flaccid paralysis (canned goods in adults, honey ingestion causing floppy baby syndrome in infants).
• Clostridium perfringens: Anaerobic rod producing alpha-toxin (lecithinase), a phospholipase that cleaves cell membranes, causing gas gangrene (myonecrosis) and double zone of hemolysis on blood agar.
• Clostridioides difficile: Anaerobic spore-former producing Toxin A (enterotoxin), which damages mucosal brush borders causing watery diarrhea, and Toxin B (cytotoxin), which induces actin depolymerization and pseudomembranous colitis following broad-spectrum antibiotic therapy (e.g., clindamycin, fluoroquinolones).

Non-Spore-Forming Rods

• Corynebacterium diphtheriae: Aerobic club-shaped rod containing metachromatic granules. Produces diphtheria toxin, which inactivates elongation factor 2 (EF-2) via ADP-ribosylation, inhibiting host protein synthesis. Causes pseudomembranous pharyngitis, bull neck lymphadenopathy, and myocarditis. Cultured on cystine-tellurite agar or L\u00f6ffler medium.
• Listeria monocytogenes: Facultative intracellular rod demonstrating tumbling motility at room temperature and actin rockets (host actin polymerization for cell-to-cell spread). Exhibits growth at cold temperatures (4\u00b0C); acquired from unpasteurized dairy products and deli meats. Causes neonatal sepsis/meningitis and meningitis in immunocompromised patients.

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Gram-Negative Cocci & Diplococci

Gram-negative cocci are predominantly represented by the genus Neisseria, which are kidney bean-shaped diplococci that are oxidase-positive.

• Neisseria meningitidis: Ferments maltose and glucose, possesses a prominent polysaccharide capsule, and is transmitted via respiratory droplets. Causes meningococcemia and meningitis, which can deteriorate into Waterhouse-Friderichsen syndrome (adrenal gland failure secondary to bilateral adrenal hemorrhage, purpura fulminans, and disseminated intravascular coagulation).
• Neisseria gonorrhoeae: Ferments glucose only (non-maltose fermenter), lacks a polysaccharide capsule, and exhibits high antigenic variation of pili. Sexually transmitted; causes urethritis, pelvic inflammatory disease (PID), Fitz-Hugh-Curtis syndrome, neonatal ophthalmia neonatorum, and septic arthritis. Cultured selectively on Thayer-Martin agar (contains vancomycin, polymyxin, and nystatin to inhibit Gram-positives, Gram-negatives, and fungi).

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Gram-Negative Bacilli & Enteric Pathogens

Gram-negative rods include Enterobacteriaceae (lactose fermenters vs non-fermenters on MacConkey agar) and non-enteric aerobes.

Enterobacteriaceae

• Escherichia coli: Lactose fermenter (pink on MacConkey agar). ETEC (enterotoxigenic) produces heat-labile (LT, increases cAMP) and heat-stable (ST, increases cGMP) toxins causing traveler's watery diarrhea. EHEC (enterohemorrhagic, strain O157:H7) produces Shiga-like toxin (verotoxin), which inactivates the 60S ribosomal subunit, causing bloody diarrhea and Hemolytic Uremic Syndrome (HUS) (triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury). EHEC does not ferment sorbitol.
• Salmonella species: Lactose non-fermenter, motile (flagella), and H2S-positive (produces black colonies on TSI agar). Salmonella Typhi causes typhoid fever (rose spots on abdomen, fever, bradycardia, abdominal pain, intestinal perforation in Peyer patches). Non-typhoidal Salmonella causes gastroenteritis associated with poultry and reptiles.
• Shigella species: Lactose non-fermenter, non-motile, low infectious dose (10-100 organisms). Produces Shiga toxin (inactivates 60S ribosomal subunit), causing bacillary dysentery (bloody diarrhea with mucus and tenesmus).
• Klebsiella pneumoniae: Lactose fermenter with a thick mucoid polysaccharide capsule. Causes severe aspiration pneumonia with currant jelly sputum in alcoholics and diabetic patients.

Non-Enteric Gram-Negative Bacilli

• Pseudomonas aeruginosa: Obligate aerobe, oxidase-positive, produces pyocyanin (blue-green pigment) and a fruity odor. Produces Exotoxin A, which inactivates EF-2 via ADP-ribosylation (identical mechanism to diphtheria toxin). Associated with ecthyma gangrenosum, pneumonia in cystic fibrosis, swimmer's ear (otitis externa), burn wound infections, and hot tub folliculitis.
• Helicobacter pylori: Curved, flagellated Gram-negative rod that is urease-positive (converts urea to ammonia and CO2, raising pH to survive gastric acid). Causes chronic gastritis, duodenal ulcers (90%), gastric ulcers, and increases risk of gastric adenocarcinoma and MALT lymphoma.

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Atypical Bacteria & Spirochetes

Mycobacteria

• Mycobacterium tuberculosis: Acid-fast bacillus stained with Ziehl-Neelsen stain due to cell wall rich in mycolic acid. Virulence factors include cord factor (trehalose dimycolate, which inhibits macrophage maturation and induces granuloma formation). Primary infection forms a Ghon complex (parenchymal lesion + hilar lymph node calcification). Histology shows caseating granulomas with central acellular necrosis, epithelioid macrophages, and Langhans giant cells.

Mycoplasma

• Mycoplasma pneumoniae: Lacks a cell wall (contains sterols in cell membrane; completely resistant to beta-lactam antibiotics). Causes walking/atypical pneumonia in young adults. Associated with elevated cold agglutinins (IgM antibodies that agglutinate RBCs at 4\u00b0C). Cultured on Eaton agar.

Spirochetes

• Treponema pallidum: Spirochete causing syphilis. Primary: painless chancre. Secondary: maculopapular rash on palms and soles, condyloma lata. Tertiary: gummas, syphilitic aortitis (tree-barking of aorta), neurosyphilis (tabes dorsalis). Screening: VDRL/RPR; Confirmation: FTA-ABS.
• Borrelia burgdorferi: Transmitted by the Ixodes tick. Causes Lyme disease. Stage 1: erythema migrans (bulls-eye rash). Stage 2: AV block, bilateral facial nerve palsy. Stage 3: migratory polyarthritis and encephalopathy.
• Leptospira interrogans: Question mark-shaped spirochete transmitted via water contaminated with animal urine. Causes Weil disease (jaundice, renal dysfunction, and hemorrhages).

Obligate Intracellular Bacteria

• Chlamydia trachomatis: Obligate intracellular organism requiring host ATP. Biphasic life cycle: Elementary body (small, infectious form that enters host cells) and Reticulate body (large, replicating form inside intracellular inclusion bodies). Visualized with Giemsa stain. Causes urethritis, PID, neonatal conjunctivitis/pneumonia, lymphogranuloma venereum (serotypes L1-L3), and reactive arthritis (Reiter syndrome: "can't see, can't pee, can't climb a tree").