18.1 Microbiology Foundations: The Organ-System TOS, Specimens & Stains, Gram-Positive and Gram-Negative Bacteria

Why this area matters

The Board of Medicine TOS for Microbiology and Parasitology has 100 items in 10 content areas, each defined by an organ system:

The competencies attached to every area are the same. You must be able to:

• classify the agent;
• describe its burden and distribution in the Philippines;
• explain its pathogenesis and clinical picture;
• choose diagnostic tests;
• correlate drugs with organisms;
• apply prevention, including immunization.

Questions therefore start from a patient: a farmer with fever and calf pain after a flood, a child with a barking cough, a woman with frothy discharge. You must work backward from the syndrome to the organism, its virulence factor, the laboratory test that confirms it and the drug or program that controls it. This section reviews the organism groups that recur across every organ system. The sections that follow apply them system by system, with Philippine programs and guidelines.

Specimens, stains and identification

• Specimens: collect before antibiotics whenever possible, from the site of infection, in the right container.
  • Blood cultures: two or more sets from separate sites.
  • Sputum: a good specimen has many neutrophils and few squamous epithelial cells.
  • Urine: clean-catch midstream.
  • CSF: processed at once.
  • Stool for ova and parasites: fresh.
• Direct examination:
  • The Gram stain classifies most bacteria.
  • The Ziehl-Neelsen or Kinyoun acid-fast stain detects mycobacteria. The modified acid-fast stain detects Nocardia and Cryptosporidium.
  • India ink shows the Cryptococcus capsule, and KOH mounts show fungal elements.
  • Giemsa stains malaria parasites, and darkfield microscopy shows spirochetes.
• Culture media:
  • blood agar shows hemolysis;
  • chocolate agar supports Haemophilus and Neisseria;
  • Thayer-Martin is selective for pathogenic Neisseria;
  • MacConkey shows lactose fermentation;
  • TCBS grows Vibrio;
  • Lowenstein-Jensen grows mycobacteria;
  • Sabouraud agar grows fungi;
  • Loeffler serum and tellurite media grow C. diphtheriae.
• Non-culture tests:
  • antigen detection, for example dengue NS1, malaria HRP-2 and pneumococcal urinary antigen;
  • serology, which needs IgM or a fourfold rise in IgG;
  • nucleic acid amplification, for example Xpert MTB/RIF and HIV viral load.

Bacterial Cell Wall Architecture & Gram Staining Principles

The Gram stain divides eubacteria into two primary categories based on cell wall composition:

• Gram-Positive Envelope: Composed of a thick, multi-layered peptidoglycan meshwork (murein) cross-linked by transpeptidases (penicillin-binding proteins, PBPs). It incorporates teichoic acid and lipoteichoic acid, which mediate adherence to mucosal surfaces and induce cytokine release (interleukin-1, tumor necrosis factor-alpha) resembling endotoxemia. The thick peptidoglycan layer traps the crystal violet-iodine complex during acetone-alcohol decolorization, staining the organism purple or deep blue.
• Gram-Negative Envelope: Possesses a thin, single-layered peptidoglycan layer enclosed within a complex outer membrane. The space between the inner and outer membranes is the periplasmic space, housing beta-lactamases, transport proteins, and oligosaccharides. The outer membrane's outer leaflet is dominated by lipopolysaccharide (LPS), composed of O-antigen polysaccharide, a conserved core polysaccharide, and Lipid A (the toxic endotoxin moiety responsible for septic shock, activating Toll-like receptor 4 [TLR4], complement cascade, and tissue factor). Acetone-alcohol decolorizes the thin wall, which subsequently takes up the counterstain safranin, staining red or pink.

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Clinically Significant Gram-Positive Cocci

Gram-positive cocci are classified initially by the catalase test ($2\text{H}_2\text{O}_2 \rightarrow 2\text{H}_2\text{O} + \text{O}_2$), differentiating catalase-positive Staphylococcus from catalase-negative Streptococcus and Enterococcus.

Molecular Basis of MRSA and VRE Resistance

• Methicillin-Resistant Staphylococcus aureus (MRSA): Resistance to all beta-lactams (penicillins, cephalosporins [except ceftaroline], carbapenems) is mediated by the chromosomal mecA gene located on the staphylococcal cassette chromosome mec (SCCmec). This gene encodes PBP2a, an altered transpeptidase with exceedingly low binding affinity for beta-lactams. Vancomycin is the drug of choice for invasive MRSA infections.
• Vancomycin-Resistant Enterococcus (VRE): Mediated by plasmid- or transposon-borne vanA or vanB gene clusters that substitute the terminal cell wall dipeptide target D-alanyl-D-alanine (D-Ala-D-Ala) with D-alanyl-D-lactate (D-Ala-D-Lac), reducing vancomycin binding affinity by more than 1,000-fold. Linezolid, daptomycin, and tigecycline are primary therapeutic alternatives.

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Clinically Significant Gram-Positive Bacilli

Gram-positive bacilli are categorized into endospore-forming and non-spore-forming genera:

Spore-Forming Gram-Positive Bacilli

• Bacillus anthracis: Aerobic spore former. Features a unique antiphagocytic poly-D-glutamate polypeptide capsule (not polysaccharide). Produces anthrax toxin consisting of three components: Protective Antigen (PA, mediates host entry), Lethal Factor (LF, a zinc-metalloprotease cleaving MAP kinase kinase, triggering necrosis and hypoxia), and Edema Factor (EF, a calmodulin-dependent adenylate cyclase that elevates intracellular cAMP). Manifests as cutaneous anthrax (painless black eschar surrounded by non-pitting gelatinous edema) or inhalational anthrax ("woolsorter's disease", hemorrhagic mediastinitis, widened mediastinum on chest radiograph).
• Bacillus cereus: Aerobic spore former. Causes food poisoning via two distinct toxins: the emetic syndrome (caused by cereulide, a preformed heat-stable enterotoxin associated with reheated fried rice, incubation 1-6 hours) and the diarrheal syndrome (caused by a heat-labile enterotoxin produced in vivo associated with contaminated meats and vegetables, incubation 8-16 hours).
• Clostridium tetani: Anaerobic spore former (tennis racket/drumstick appearance). Spores in soil enter wounds. Produces tetanospasmin, an A-B neurotoxin that travels via retrograde axonal transport to inhibitory Renshaw interneurons in the spinal cord. Its zinc-metalloprotease cleaves synaptobrevin (VAMP), a SNARE protein, preventing release of inhibitory neurotransmitters GABA and glycine. This disinhibits lower motor neurons, causing severe spastic paralysis, trismus (lockjaw), risus sardonicus (sardonic smile), and opisthotonos (hyperextended arching of the spine).
• Clostridium botulinum: Anaerobic spore former. Produces botulinum neurotoxin, which cleaves SNARE proteins (SNAP-25, synaptobrevin) in presynaptic peripheral cholinergic nerve terminals, preventing acetylcholine (ACh) release at neuromuscular junctions. This results in symmetric, descending flaccid paralysis, diplopia, dysarthria, dysphagia, and respiratory arrest. Infant botulism occurs from ingesting spores in unpasteurized honey, manifesting as constipation and hypotonia ("floppy baby syndrome").
• Clostridioides difficile: Anaerobic spore former. Proliferates following broad-spectrum antibiotic therapy (clindamycin, cephalosporins, fluoroquinolones). Produces Toxin A (enterotoxin), which damages brush-border enterocytes and incites inflammation, and Toxin B (cytotoxin), which depolymerizes actin filaments, causing mucosal necrosis and pseudomembrane formation (pseudomembranous colitis). Diagnosis is made by stool nucleic acid amplification or toxin enzyme immunoassay. Treatment involves oral vancomycin or oral fidaxomicin.
• Clostridium perfringens: Anaerobic spore former. Produces alpha-toxin (lecithinase / phospholipase C), which hydrolyzes cellular phospholipids, causing massive hemolysis, vascular destruction, and tissue necrosis (gas gangrene / clostridial myonecrosis with crepitus).

Non-Spore-Forming Gram-Positive Bacilli

• Corynebacterium diphtheriae: Club-shaped pleomorphic Gram-positive rod in Chinese-character or palisade formations. Produces diphtheria toxin, an A-B exotoxin whose catalytic A domain catalyzes the ADP-ribosylation of elongation factor 2 (EF-2), terminating host protein synthesis. Causes pharyngitis with an adherent, thick, gray-white pseudomembrane on the tonsils and posterior pharynx; attempts to dislodge it cause bleeding. Systemic toxin absorption causes myocarditis, cardiac arrhythmias, and motor neuropathies (recurrent laryngeal nerve palsy). Cultured on Loeffler coagulated serum slant and potassium tellurite agar (black/gray colonies); toxigenicity is confirmed via the Elek immunodiffusion test.
• Listeria monocytogenes: Facultative intracellular Gram-positive rod. Exhibits distinctive tumbling motility at room temperature (20-25°C) and cold enrichment growth at 4°C. Spreads from cell to cell by polymerizing host actin into "actin rockets" mediated by the ActA protein. Transmitted via unpasteurized milk, soft cheeses, and contaminated deli meats. In neonates and elderly or immunocompromised hosts, it causes life-threatening meningitis and sepsis. Listerial meningitis requires ampicillin (which possesses activity against Listeria, unlike cephalosporins, to which Listeria exhibits intrinsic resistance).

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Clinically Significant Gram-Negative Cocci

The genus Neisseria comprises Gram-negative diplococci that are oxidase-positive and grow on enriched chocolate agar or selective Thayer-Martin medium (containing vancomycin, colistin, nystatin, and trimethoprim to inhibit Gram-positives, Gram-negative bacilli, fungi, and Proteus swarming).

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Gram-Negative Bacilli: Enteric & Non-Enteric Pathogens

Gram-negative bacilli are initially differentiated by their ability to ferment lactose on MacConkey agar (pink colonies = fermenters; colorless colonies = non-fermenters):

Lactose Fermenters (Pink on MacConkey Agar)

• Escherichia coli: Indole-positive, metallic green sheen on Eosin Methylene Blue (EMB) agar.
  • Enterotoxigenic E. coli (ETEC): Produces Heat-Labile toxin (LT), which activates adenylate cyclase ($cAMP \uparrow$), and Heat-Stable toxin (ST), which activates guanylate cyclase ($cGMP \uparrow$). Causes watery traveler's diarrhea.
  • Enterohemorrhagic E. coli (EHEC / STEC, prototype O157:H7): Does not ferment sorbitol on sorbitol-MacConkey agar (SMAC). Produces Shiga-like toxins (Stx1 and Stx2), which inactivate the 60S ribosomal subunit by cleaving 28S rRNA. Causes hemorrhagic colitis and Hemolytic Uremic Syndrome (HUS): a clinical triad of microangiopathic hemolytic anemia (schistocytes on blood smear), thrombocytopenia, and acute kidney injury. Antibiotic therapy is contraindicated in STEC infections, as it triggers bacteriophage induction and increases toxin release, accelerating HUS progression.
  • Uropathogenic E. coli (UPEC): Possesses P-fimbriae (pyelonephritis-associated pili) that bind galactose receptors on uroepithelial cells; accounts for > 80% of uncomplicated community-acquired UTIs.
• Klebsiella pneumoniae: Non-motile, prominent polysaccharide mucoid capsule. Ferments lactose vigorously. Causes community-acquired lobar pneumonia in alcoholics and diabetics, characteristically producing thick, bloody, "currant jelly" sputum with upper lobe cavitary necrosis.

Non-Lactose Fermenters (Colorless on MacConkey Agar)

• Salmonella enterica serovar Typhi: Oxidase negative, produces $\text{H}_2\text{S}$ (black colonies on Hektoen enteric agar), motile. Transmitted via contaminated water and food. Penetrates intestinal M-cells of Peyer patches and disseminates inside macrophages. Manifests as typhoid (enteric) fever: step-ladder fever, relative bradycardia (Faget sign), abdominal pain, hepatosplenomegaly, faint erythematous "rose spots" on the trunk, and leukopenia. Asymptomatic carrier state is maintained by chronic colonization in the gallbladder. First-line treatment includes ceftriaxone or ciprofloxacin.
• Shigella dysenteriae & S. flexneri: Oxidase negative, non-motile, does NOT produce $\text{H}_2\text{S}$. Exhibits an exceedingly low infectious inoculum (as few as 10-100 organisms cause disease). Invades intestinal M-cells and escapes the phagosome to polymerize actin for intra- and intercellular motility. Causes bacillary dysentery: fever, severe tenesmus, and frequent small-volume bloody, mucoid stools.
• Pseudomonas aeruginosa: Obligate aerobe, oxidase-positive, non-lactose fermenter. Produces the blue-green pigments pyocyanin and pyoverdine, and cultures have a characteristic sweet, grape-like odor. Possesses Exotoxin A, which inhibits host protein synthesis through ADP-ribosylation of EF-2 (identical mechanism to diphtheria toxin). Important opportunistic pathogen causing ventilator-associated pneumonia, ecthyma gangrenosum (black necrotic skin bullae in neutropenic patients), hot tub folliculitis, malignant otitis externa in diabetics, and corneal ulcers in contact lens wearers.
• Vibrio cholerae: Comma-shaped, oxidase-positive, grows on TCBS (thiosulfate-citrate-bile salts-sucrose) agar as yellow colonies. Thrives in brackish coastal waters and contaminated water supplies. Produces cholera toxin, an A-B enterotoxin whose A subunit ADP-ribosylates the $G_{\alpha s}$ regulatory protein, maintaining adenylate cyclase in a continuously active state ($cAMP \uparrow$). This leads to active secretion of chloride and bicarbonate ions into the intestinal lumen, causing massive, painless, watery "rice-water" diarrhea that can exceed 1 liter/hour, precipitating lethal hypovolemic shock. Primary therapy is aggressive fluid and electrolyte replacement using Oral Rehydration Salts (ORS) or IV Ringer's lactate; doxycycline shortens shedding.
• Campylobacter jejuni: Curved, comma- or gull-wing-shaped Gram-negative rod, oxidase-positive, microaerophilic, grows at 42°C on Campy-BAP agar. Transmitted by undercooked poultry and unpasteurized milk. Causes inflammatory, bloody diarrhea and serves as the leading antecedent infectious trigger for Guillain-Barré syndrome (via molecular mimicry between lipooligosaccharides and human peripheral nerve gangliosides GM1) and reactive arthritis.
• Helicobacter pylori: Curved, flagellated Gram-negative rod. Produces abundant urease, which hydrolyzes gastric urea into ammonia and carbon dioxide, neutralizing local gastric acidity. Causes chronic active gastritis, duodenal and gastric peptic ulcers, and elevates the risk for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma.
• Haemophilus influenzae: Pleomorphic Gram-negative coccobacillus. Fastidious organism requiring Factor X (hemin) and Factor V (NAD) for growth on chocolate agar. Exhibits the satellite phenomenon, growing as tiny colonies around Staphylococcus aureus streaks on sheep blood agar (because S. aureus lyses RBCs to liberate Factor X and actively secretes Factor V). Encapsulated type b (Hib) strains cause acute epiglottitis ("thumbprint sign" on lateral neck radiograph), pediatric meningitis, and septic arthritis.

Exotoxins and endotoxin at a glance

Viruses are classified by nucleic acid and envelope. Fungi are classified as yeasts, molds or dimorphic fungi. Their individual diseases are covered with the organ systems they affect.