3.2 Gram-Positive Cocci & Bacilli

Gram-Positive Cocci & Bacilli

Gram-positive organisms retain the primary crystal violet stain and appear a deep purple under the microscope. This group includes a vast array of pathogens responsible for everything from simple skin infections to life-threatening sepsis, meningitis, and toxic shock. The initial identification pathways for Gram-positive cocci (GPC) rely heavily on a few rapid, highly specific biochemical tests, while Gram-positive bacilli (GPB) require evaluation of spore formation, motility, and specific metabolic profiles.

The Catalase Test: The First Branching Point

The most important initial diagnostic test for any newly isolated GPC is the catalase test. The enzyme catalase converts toxic hydrogen peroxide (H2O2)—a byproduct of aerobic metabolism—into water and oxygen gas.

• Catalase Positive: A robust evolution of oxygen bubbles occurs immediately upon the addition of 3% H2O2. This definitively indicates the organism belongs to the Staphylococcus or Micrococcus genera (which generally appear in grapelike clusters).
• Catalase Negative: No bubbles form. This points to the Streptococcus or Enterococcus genera (which classically appear in pairs or chains).

Catalase-Positive GPC: The Staphylococci

Once an organism is confirmed as a catalase-positive GPC, the next definitive, crucial step is the coagulase test. Coagulase is an enzyme that causes blood plasma to clot by converting fibrinogen to fibrin.

Staphylococcus aureus

S. aureus is a formidable major human pathogen responsible for furuncles, carbuncles, impetigo, toxic shock syndrome (via TSST-1), food poisoning (via preformed enterotoxins), osteomyelitis, and bacteremia. It is coagulase-positive.

• Testing Methods: The slide coagulase test detects "bound coagulase" (clumping factor) directly on the cell wall, causing rapid agglutination. The tube coagulase test detects extracellular "free coagulase" which forms a solid clot in rabbit plasma after 4-24 hours of incubation. Modern labs often use rapid latex agglutination tests that detect both clumping factor and S. aureus Protein A simultaneously.
• Morphology: It typically produces medium to large, smooth, distinctly golden, or cream-colored colonies on BAP and is usually strongly beta-hemolytic.

Coagulase-Negative Staphylococci (CoNS)

Organisms that are catalase-positive but coagulase-negative are collectively grouped as CoNS. While frequently normal skin flora and common blood culture contaminants, they are significant opportunistic pathogens.

• Staphylococcus epidermidis: A ubiquitous skin colonizer. It is highly notorious for producing an extracellular polysaccharide slime layer (biofilm) that allows it to adhere tenaciously to indwelling medical devices (intravascular catheters, prosthetic heart valves, orthopedic hardware). It is usually non-hemolytic and is susceptible to the antibiotic novobiocin.
• Staphylococcus saprophyticus: A very common cause of urinary tract infections, second only to E. coli in young, sexually active women. It is distinctively resistant to novobiocin, a key diagnostic feature separating it from other CoNS in urine cultures.
• Staphylococcus lugdunensis: A highly virulent CoNS that can cause destructive endocarditis mimicking S. aureus. It can occasionally give a positive slide coagulase test (clumping factor positive) but is tube coagulase negative. It is definitively identified by being strongly positive for both PYR (L-pyrrolidonyl arylamidase) and ornithine decarboxylase.

Catalase-Negative GPC: The Streptococci and Enterococci

The classification and identification of Streptococci rely heavily on their hemolytic patterns on 5% sheep blood agar, followed by Lancefield serogrouping (based on cell wall C carbohydrates) and specific biochemical reactions.

Beta-Hemolytic Streptococci

These organisms produce hemolysins (Streptolysin O and S) that completely lyse red blood cells, creating a clear, transparent zone around the colony.

• Group A Streptococcus (S. pyogenes): Causes strep throat (pharyngitis), scarlet fever, impetigo, and severe necrotizing fasciitis. Crucially, untreated infections can lead to severe post-streptococcal sequelae, including acute rheumatic fever and post-streptococcal glomerulonephritis. It is universally susceptible to bacitracin (A disk) and is PYR positive.
• Group B Streptococcus (S. agalactiae): A significant cause of devastating neonatal sepsis and meningitis, typically acquired during vaginal delivery. It is resistant to bacitracin. Diagnostically, it is positive for the CAMP test (producing an arrowhead of enhanced beta-hemolysis when grown near S. aureus) and is positive for hippurate hydrolysis.

Alpha-Hemolytic Streptococci

These organisms produce hydrogen peroxide that oxidizes hemoglobin to methemoglobin, creating a partial, greenish discoloration of the agar.

• Streptococcus pneumoniae: Causes community-acquired pneumonia, adult meningitis, and pediatric otitis media. It possesses a thick, antiphagocytic polysaccharide capsule (the primary virulence factor) and typically appears as lancet-shaped Gram-positive diplococci. It is uniquely susceptible to optochin (P disk) and is rapidly lysed by bile salts (bile soluble).
• Viridans Group Streptococci: A large group (e.g., S. mutans, S. mitis) that are normal flora of the human oral cavity and GI tract. They are a major cause of subacute bacterial endocarditis following dental procedures. Unlike S. pneumoniae, they are resistant to optochin and bile insoluble.

Gamma-Hemolytic (Non-Hemolytic) Streptococci and Enterococci

• Enterococcus species (E. faecalis, E. faecium): Common causes of UTIs, intra-abdominal infections, and healthcare-associated endocarditis. They are incredibly robust, demonstrating the ability to grow in 6.5% NaCl broth and the ability to hydrolyze esculin in the presence of bile (Bile Esculin positive). They are also PYR positive.
• Group D Streptococci (e.g., S. bovis / S. gallolyticus): Also Bile Esculin positive, but critically, they cannot grow in 6.5% NaCl and are PYR negative. The isolation of S. bovis from blood cultures is a classic clinical pearl strongly associated with hidden gastrointestinal malignancies (colon cancer).

Gram-Positive Bacilli (GPB)

Gram-positive bacilli encompass a highly diverse group of organisms, ranging from environmental spore-formers to dangerous intracellular pathogens.

• Bacillus species: Large, boxcar-shaped, aerobic or facultatively anaerobic spore-forming GPB.
  • Bacillus anthracis: The causative agent of anthrax. Diagnostically, it is uniquely non-motile and non-hemolytic on BAP, often displaying a classic "Medusa head" colony morphology.
  • Bacillus cereus: A common cause of food poisoning (often associated with fried rice). Unlike B. anthracis, it is highly motile and broadly beta-hemolytic.
• Listeria monocytogenes: A small, non-spore-forming GPB that causes severe foodborne illness (listeriosis), leading to devastating outcomes (stillbirth, meningitis) in pregnant women and immunocompromised individuals. It is uniquely capable of robust growth at refrigeration temperatures (cold enrichment). It exhibits distinctive "tumbling" motility on wet mount at room temperature (25°C) and an "umbrella" pattern of motility in semi-solid agar. It is also CAMP test positive (producing a block-shaped zone of hemolysis).
• Corynebacterium species: Club-shaped, highly pleomorphic GPB that frequently arrange in "V" or "L" shapes, described as "palisades" or "Chinese letters."
  • Corynebacterium diphtheriae: The primary pathogen, causing the respiratory disease diphtheria via the production of a potent exotoxin. Toxigenicity must be confirmed, historically via the in vitro Elek immunodiffusion test. It is often isolated on specialized selective media like Tinsdale agar (forming black colonies with a brown halo) or Loeffler medium.
  • Other species are commonly termed "diphtheroids" and are generally considered normal skin flora or contaminants, though they can cause infection in immunocompromised hosts.
• Erysipelothrix rhusiopathiae: Causes erysipeloid, a localized skin infection often occupational (butchers, fish handlers). It is unique among non-spore-forming GPB as it produces significant H2S on TSI agar.
• Nocardia species: Aerobic, branching, beaded Gram-positive bacilli that are uniquely partially acid-fast. They can cause severe pulmonary and cutaneous infections, particularly in immunocompromised individuals. Growth can take weeks to appear, often resembling dry, chalky, or velvety molds.