Bacterial structure, classification and virulence factors

At a glance

• The cell envelope decides the Gram stain result and which antibiotics can reach their target. A Gram stain is often the first result a physician acts on.
• Gram-positive bacteria have a thick peptidoglycan wall and no outer membrane. Gram-negative bacteria have a thin wall and an outer membrane containing lipopolysaccharide (LPS), which drives septic shock.
• Some bacteria do not Gram stain usefully: mycobacteria (waxy mycolic acid wall, acid-fast), Mycoplasma (no wall), and obligate intracellular organisms. These need different tests and drugs. See intracellular bacteria, mycobacteria and spirochaetes.
• Virulence factors explain who gets sick and how: capsules (asplenia, complement deficiency), biofilm (device infections), and toxins (toxic shock, tetanus, C. difficile).
• In toxin-mediated disease the patient can stay unwell after the organism is dead. Management adds toxin suppression, toxin neutralisation and source control to killing.

The envelope and why it matters for drugs

Feature	Gram-positive	Gram-negative	Clinical consequence
Peptidoglycan	Thick, outer layer	Thin, between two membranes	Both are beta-lactam targets
Outer membrane	Absent	Present, with porins	Excludes vancomycin; porin loss adds to carbapenem resistance
LPS (endotoxin)	Absent	Lipid A in outer membrane	Potent trigger of septic shock and DIC
Periplasmic space	No outer-membrane-bounded periplasm; beta-lactamases secreted	Present	Concentrates beta-lactamases near their target
Gram stain	Purple	Pink	Guides empiric therapy within hours

• Vancomycin binds the D-Ala-D-Ala end of peptidoglycan precursors. It is too large to cross the outer membrane, so it covers Gram-positives only.
• Daptomycin inserts into the Gram-positive cytoplasmic membrane. It is inactivated by pulmonary surfactant, so it fails in pneumonia.
• Aminoglycosides need oxygen-dependent transport into the cell. Anaerobes and acidic pus defeat them.
• Polymyxins (colistin) bind lipid A, which is why they work only on Gram-negatives.

More detail is in beta-lactam pharmacology and aminoglycoside and glycopeptide pharmacology.

Reading the Gram stain

Gram stain	Likely organisms	Usual empiric reasoning
Gram-positive cocci in clusters	Staphylococcus aureus, coagulase-negative staphylococci	Flucloxacillin or cefazolin; vancomycin if MRSA risk
Gram-positive cocci in chains	Streptococci, enterococci	Benzylpenicillin for strep; amoxicillin for E. faecalis
Gram-positive diplococci, lancet shaped	Streptococcus pneumoniae	Benzylpenicillin or ceftriaxone, by site and MIC
Gram-positive bacilli	Listeria, Corynebacterium, Bacillus, Clostridium, Cutibacterium	Context: CSF in older adult means Listeria
Gram-negative diplococci	Neisseria, Moraxella	Ceftriaxone for meningococcus or gonococcus
Gram-negative bacilli	Enterobacterales, Pseudomonas	Ceftriaxone or gentamicin; antipseudomonal agent if risk
Gram-negative coccobacilli	Haemophilus, Acinetobacter	Acinetobacter may need a carbapenem

Worked example: one positive blood culture bottle

A 58-year-old man on haemodialysis has fever and rigors. At 14 hours one aerobic bottle flags with Gram-positive cocci in clusters.

• Clusters mean Staphylococcus. In a dialysis patient with a line, S. aureus must be assumed until identification, because it carries about 20 to 30% mortality and needs at least 14 days of therapy (IV, or early oral step-down after 5 to 7 days in selected uncomplicated cases, as in the SABATO trial), echocardiography and line removal.
• He has healthcare exposure, so start vancomycin (MRSA risk) and add cefazolin or flucloxacillin until susceptibility returns. Vancomycin alone kills MSSA more slowly than a beta-lactam.
• A single bottle of coagulase-negative staphylococci with a long time to positivity (for example more than 24 hours) in only one of two sets is more often a skin contaminant. Rapid PCR identification (for example mecA and nuc) shortens this uncertainty to an hour or two. See culture and susceptibility testing and bacteraemia.

Pitfall: Gram-positive bacilli in blood are often dismissed as diphtheroid contaminants. In an older, pregnant or immunosuppressed patient, consider Listeria until proven otherwise.

Oxygen requirements

• Obligate anaerobes (Bacteroides fragilis, Clostridium, Fusobacterium, Prevotella) live below the diaphragm and in the mouth. They cause abscesses, foul-smelling pus and gas in tissue.
• Anaerobes lack the oxygen-dependent uptake that aminoglycosides need. Cover them with metronidazole, a beta-lactam/beta-lactamase inhibitor or a carbapenem.
• Lemierre syndrome (Fusobacterium necrophorum septic jugular thrombophlebitis) follows pharyngitis in young adults and seeds the lungs.

Virulence factors at the bedside

Capsules

• A polysaccharide capsule resists phagocytosis until antibody and complement opsonise it. The spleen clears opsonised bacteria and makes antibody to polysaccharide antigens.
• Asplenia or hyposplenism (splenectomy, sickle cell disease, coeliac disease) predisposes to fulminant pneumococcal, meningococcal and Hib sepsis. Lifelong risk. Give a single dose of 21vPCV (2026 NIP schedule), MenACWY, MenB, Hib and annual influenza vaccine; daily prophylactic amoxicillin or phenoxymethylpenicillin, for a period after splenectomy that depends on age and ongoing risk (often at least the first few years, longer if high risk; follow the Immunisation Handbook and local guidance); a standby emergency course; and education.
• Terminal complement (C5 to C9) deficiency and eculizumab or ravulizumab give recurrent or severe Neisseria infection. See complement.
• Polysaccharide antigens alone give a T-cell-independent response that is poor under 2 years and lacks memory. Conjugate vaccines link the polysaccharide to a protein carrier. See vaccine immunology.

Adhesion and biofilm

• Staphylococci (especially S. epidermidis and S. aureus), Pseudomonas and Candida form biofilm on lines, valves and joints.
• Bacteria in biofilm grow slowly and sit behind a matrix, so MICs measured in the laboratory overstate the effect in the patient.
• Consequences: device removal is usually needed for cure, and rifampicin is added for staphylococcal prosthetic joint infection retained after debridement. See device infections.

Endotoxin

• LPS from Gram-negative bacteria triggers TLR4 on macrophages, with cytokine release, vasodilatation, capillary leak and DIC. See innate immunity.
• Rapid bacterial lysis after the first antibiotic dose can briefly worsen the inflammatory response. This is not a reason to delay antibiotics.

Toxin-mediated syndromes: why management differs

Some diseases are caused by a toxin released from a localised or even trivial focus. The organism may not be found in blood, and killing it does not reverse toxin already bound to tissue.

Syndrome	Organism and typical focus	What changes in management
Streptococcal toxic shock	Group A strep; necrotising fasciitis, cellulitis, postpartum	Urgent surgical debridement, benzylpenicillin plus clindamycin, IVIG in severe disease; chemoprophylaxis for close household contacts
Staphylococcal toxic shock	S. aureus; tampons, packing, wounds, surgical sites	Remove the focus, anti-staphylococcal beta-lactam (or vancomycin) plus clindamycin; blood cultures often negative
Tetanus	Clostridium tetani; contaminated wound	Tetanus immunoglobulin early, wound debridement, metronidazole, benzodiazepines and airway support; vaccinate on recovery
Botulism	Clostridium botulinum; food, wounds, injecting drug use	Antitoxin early (neutralises circulating toxin only), ventilatory support; notify public health urgently
Diphtheria	Corynebacterium diphtheriae; pharynx or skin	Diphtheria antitoxin plus antibiotics; check for myocarditis
C. difficile infection	Colon after antibiotics	Stop the inciting antibiotic, oral vancomycin or fidaxomicin; toxin assay separates disease from colonisation

• Inoculum effect. In a large, slowly dividing bacterial population, penicillin-binding protein expression falls and beta-lactams lose activity. Clindamycin works on the ribosome regardless of growth phase, which is why it is added in necrotising group A strep infection.
• Antitoxin timing. Tetanus and botulinum antitoxins neutralise only toxin not yet bound to nerve terminals. Give them on clinical suspicion; do not wait for confirmation.
• No immunity after disease. The amount of tetanus toxin that causes disease is too small to immunise. Recovered patients still need a full primary course.
• Laboratory. For C. difficile, a positive PCR or GDH antigen with a negative toxin result often means colonisation. Interpret with the stool frequency and clinical picture.

See sepsis and cellulitis and bacterial skin infections for clinical management.

How it's tested

• A blood or CSF Gram stain result, asking for the most appropriate empiric antibiotic or the likely organism.
• Explaining why a drug fails: vancomycin for E. coli, beta-lactams for Mycoplasma, daptomycin for pneumonia, gentamicin for an abscess.
• A patient with asplenia, complement deficiency or on eculizumab presenting with sepsis.
• A toxic shock or tetanus vignette asking which additional therapy is needed beyond a bactericidal antibiotic.
• Device infections that relapse despite susceptible organisms.

Pitfalls

• Pitfall: treating MSSA bacteraemia with vancomycin alone once susceptibility is known. Switch to flucloxacillin or cefazolin.
• Pitfall: assuming a negative blood culture excludes staphylococcal toxic shock. Most cases are bacteraemia-negative.
• Pitfall: withholding tetanus immunoglobulin until the diagnosis is confirmed. It cannot reverse toxin already bound.
• Pitfall: a "sterile" Gram stain in atypical pneumonia. Mycoplasma and Legionella are not seen on routine Gram stain.
• Pitfall: following the pre-2026 pneumococcal sequence (13vPCV then 23vPPV) in asplenia. Adults now get a single dose of 21vPCV, plus MenACWY, MenB, Hib and annual influenza.