Gram-negative

**Gram stain principle:** Crystal violet (primary stain) → iodine (mordant, forms crystal violet-iodine complex) → alcohol/acetone decolorizer → safranin counterstain. Gram-positive organisms have a thick peptidoglycan wall that retains the complex (purple). Gram-negative organisms have a thin peptidoglycan layer; the decolorizer also disrupts the outer membrane, so the complex washes out and the cells stain pink/red.

**Cell envelope structure (outside → in):** - Outer membrane: phospholipid inner leaflet, LPS outer leaflet, porins - Periplasmic space: contains peptidoglycan (thin), β-lactamases, and other enzymes - Inner (cytoplasmic) membrane - Gram-negatives have NO teichoic or lipoteichoic acids (these are gram-positive features).

**LPS (endotoxin):** - O antigen (outermost polysaccharide; most variable, basis of serotyping) - Core polysaccharide - Lipid A (the toxic component) - Lipid A binds the CD14/TLR4/MD-2 complex on macrophages. This triggers release of IL-1, IL-6, and TNF-α, causing fever, hypotension, DIC, and septic shock. It also activates the alternative complement pathway and Hageman factor (XII).

**Clinical significance:** - The outer membrane limits entry of many drugs. Penicillin G and vancomycin are generally ineffective, and vancomycin is too large to cross porins. Extended-spectrum penicillins (piperacillin), 3rd/4th-generation cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and aztreonam (gram-negative aerobes only) cover them. - Resistance mechanisms: ESBLs, carbapenemases (e.g., KPC), AmpC, efflux pumps, porin loss, plasmid-mediated transfer (conjugation). - Antibiotic-induced lysis can release endotoxin, causing a Jarisch-Herxheimer-like worsening in some settings.

**Major gram-negative groups (high-yield):** - **Cocci:** Neisseria meningitidis and N. gonorrhoeae (diplococci), Moraxella catarrhalis - **Coccobacilli:** Haemophilus influenzae, Bordetella pertussis, Acinetobacter, Brucella, Francisella, Pasteurella - **Lactose fermenters (pink on MacConkey):** E. coli, Klebsiella, Enterobacter, Citrobacter - **Non-lactose fermenters:** Salmonella, Shigella, Proteus, Pseudomonas (oxidase +), Yersinia - **Curved/spiral:** Vibrio, Campylobacter, Helicobacter - **Others:** Legionella (stains poorly; use silver stain or BCYE agar), Bacteroides (anaerobes)

**Gram-negative organisms that don't Gram stain well:** - Rickettsia (Giemsa), Chlamydia (no muramic acid; Giemsa), Mycoplasma (no cell wall; sterol-containing membrane), Legionella (silver stain), Treponema (dark-field/silver), Bartonella (Warthin-Starry silver), Coxiella.

**Gram-negative sepsis:** - Most common causes: E. coli (UTI, intra-abdominal), Klebsiella, Pseudomonas (nosocomial, burns, neutropenia, cystic fibrosis), Neisseria meningitidis (meningococcemia with Waterhouse-Friderichsen syndrome). - Treatment is prompt antibiotics, fluids, and vasopressors.

**Exam pearls:** - Thin peptidoglycan + outer membrane + LPS = gram-negative. - Gram-positive bacteria produce exotoxins, whereas gram-negatives are classically associated with endotoxin (exceptions: Vibrio cholerae, ETEC, Shigella, EHEC, Bordetella, and Yersinia pestis all produce exotoxins). - The periplasmic space of gram-negative bacteria is where β-lactamases are located (in gram-positives, they are secreted).