Streptococci vs Staphylococci

• Catalase negative, Gram-positive cocci that appear in pairs and chains include Streptococcus, Enterococcus, and Strep-like organisms.

Characteristics

• These Gram-positive cocci (GPC) are found in chains or pairs.
• They are catalase negative, which distinguishes them from staphylococci.
• These organisms are facultative anaerobes.
• Some are capnophilic, requiring increased CO2.
• They can grow on BAP, CHOC, CNA, and PEA.
• BAP is preferred for hemolysis demonstration.

Cell Wall Structure

• Has a typical gram-positive cell wall with peptidoglycan and teichoic acid.
• Capsules can be present in young cultures.
• C (capsular) polysaccharide antigen is present.

Classification Schemes

• The same organism can have multiple names due to the different classification systems.
• There are four classification systems: hemolytic pattern, serologic grouping, common names, and physiologic characteristics.

Hemolytic Pattern

• Exotoxins damage red blood cells (RBCs).
• Hemolysis is observed on Blood Agar Plates (BAP).
• Alpha hemolysis appears green and incomplete due to hemoglobin reduction to methemoglobin, causing a green discoloration.
• Alpha prime hemolysis has narrow alpha hemolysis with wide beta hemolysis or a small zone of complete hemolysis surrounded by partial lysis.
• Beta hemolysis is clear and complete.
• Gamma hemolysis exhibits no hemolysis.

Serogrouping

• Lancefield grouping is based on cell-wall antigens (C carbohydrate).
• Latex agglutination techniques are commercially available for grouping.
• Not all streptococci can be identified through serogrouping, but includes groups A, B, C, D, F, and G.

Common Names

• Includes β strep or group A strep, Group B strep, Group D strep, α strep, Pneumococci, Viridance group, and Enterococcus.

Physiologic Properties

• Classification based on biochemical reactions, with each organism receiving a genus and species name.

S. pyogenes (virulence)

• Hemolysis occurs due to two toxins: Streptolysin S (SLS) and Streptolysin O (SLO).
• Streptolysin S (SLS) is oxygen stable.
• Streptolysin O (SLO) is oxygen labile.
• Hemolysis from SLO may not be detected if incubated aerobically.
• SLO is protected from oxygen when BAP is stabbed or cut, creating a localized anaerobic environment.
• SLO also lyses white blood cells (WBCs) and platelets.
• SLO stimulates the production of anti-streptolysin O (ASO).
• Lipoteichoic acid and fibronectin-binding protein contribute to adhesion.
• M protein is needed for virulence, resists phagocytosis, and increases adherence to mucosal surfaces.
• Erythrogenic/pyogenic toxins can cause Streptococcus toxic shock syndrome (STSS), similar to TSST-1, leading to fever, rash, and organ damage.
• Streptokinase lyses blood clots (RBC, platelets, WBCs).

S. pyogenes Diseases

• Pharyngitis (strep throat).
• Scarlet fever.
• Streptococcal toxic shock syndrome.
• Skin or pyodermal infections.
• Post-streptococcal sequelae including rheumatic fever and acute glomerulonephritis.

Pharyngitis/Tonsillitis

• Strep throat is a common S. pyogenes disease mostly seen in children.
• Rapid detection kits (latex) are available.

Pharyngitis Testing

• Antigen detection tests offer an alternative to throat culture and are fast.
• Commercially available kits have high specificity but low sensitivity (60-95%).
• Follow up culturing needed following negative results .

Scarlet Fever and Streptococcal TSS

• Scarlet fever involves a streptococcal pyogenic exotoxin, leading to fever with a rash after a bacterial infection.
• Streptococcal toxic shock syndrome (STSS) can cause organ shutdown and death, mainly due to erythrogenic/pyogenic toxins.

Pyodermal (Skin) Infections

• Impetigo
• Erysipelas which is an acute infection of the upper dermis and superficial lymphatics.
• Necrotizing Fasciitis - flesh eating bacteria

Post-Streptococcal Sequelae

• These follow S. pyogenes infections and are autoimmune in origin.
• Acute rheumatic fever after pharyngitis causes fever, inflammation, and rheumatic heart disease.
• Acute glomerulonephritis can occur after pharyngitis or pyoderma.

Streptococcus pyogenes Identification

• Group A strep, belongs to the group ẞ Strep.
• It is not part of normal flora (NF), but can be asymptomatic in young children.
• Colonies are small (less than 1 mm) and translucent.
• β hemolytic.
• Colonies are small and pinpoint with a wide zone of hemolysis.
• Hemolysis is improved if BAP is stabbed, cut, or incubated anaerobically.

Identification

• Direct latex agglutination test is implemented.
• Culture on Blood Agar Plate (BAP) shows β-hemolytic colonies.
• Serogrouping is done using Lancefield group A.
• PYR (L-pyrrolidonyl arylamidase) test result is positive.
• Bacitracin (A) disk susceptible.
• SXT (sulfamethoxazole -trimethoprim) disk resistant.
• Traditional serotyping (M-T-R) includes M protein, T (Lancefield group), and R protein
• Current typing method uses sequencing of the M protein gene.

Bacitracin (A) Disk Susceptibility

• The test should only be done on beta hemolytic streptococci and is inhibited by bacitracin.
• The A disk is used to identify group A streptococci.
• Group A strep is susceptible to the A disk, while Group B strep shows resistance.

Trimethoprim-Sulfamethoxazole (SXT)

• This test uses two antibiotics.
• Groups A and B are resistant, while groups C, F, and G are susceptible.
• Groups C and G, called S. dysgalactiae, can cause disease similar to Group A.

PYR Hydrolysis Test

• This test detects PYRase.
• L-pyrrolidonyl-β-naphthylamide (PYR) is hydrolyzed to dimethylaminocinnamaldehyde.
• The test is positive for Enterococcus, Group A, and other organisms.
• Organisms with L-pyrrolidonyl arylamidase can hydrolyze L-pyrrolidonyl-β-naphthylamide to release L-pyrrolidone.
• Visual detection is achieved by adding PYR/LAP Reagent, where p-dimethylaminocinnamaldehyde combines with β-naphthylamide to form a red Schiff base, changing the reaction from yellow to red color.

S. agalactiae

• Belongs to Group B Streptococci.
• It is part of the normal flora of the genitourinary tract (25% carrier rate).
• Has a polysaccharide capsule.
• Is extremely dangerous to infants.
• Group B streptococci causes serious infection in Babies.

Hemolysis and Colonial Morphology

• Exhibits grayish-white, flat, creamy colonies (>1mm).
• Most are beta hemolytic, with larger colonies and a very small zone of hemolysis.

Identification

• Serogrouping utilizes Lancefield group B.
• Bacitracin (A) disk resistant.
• CAMP positive.
• Hippurate hydrolysis positive.
• Molecular analysis is completed.

Antigen Detection Tests

• Commercial tests using latex agglutination.
• It detects antigen in CSF, serum, urine, and vaginal secretions.
• In urine and vaginal specimens, NF may cause false positives.

CAMP Test

• S. aureus produces β-lysin and group B streptococci produces CAMP factor.
• Enhanced hemolysis is observed when CAMP factor and β-lysin act synergistically on BAP.
• A test organism is streaked perpendicular to S. aureus without them touching
• Arrowhead-shaped hemolysis at junction of two streaks is a positive result.

Hippurate Hydrolysis

• Detects hippuricase.
• Sodium hippurate (N-benzoylglycine) is hydrolyzed into sodium benzoate and glycine.
• Ninhydrin reacts with glycine to form a purple color.
• A 2-hour rapid test is available.
• Additional organisms test positive.

Molecular Detection

• Beta strep screen of pregnant women.
• Group B strep screen utilizing real time PCR has a significantly higher recovery rate (98%) than culture (75%).

Clinical Significance

• Neonatal sepsis and meningitis can occur due to the organism being vaginal NF, colonizing infants during birth; few develop disease however.
• UTI in adults.
• Skin and soft tissue infections.
• Postpartum (after birth) fever.
• Sepsis.

β-Hemolytic Streptococci (Groups C, F and G)

• Considered part of normal flora in the skin, nasopharynx, GI tract, and GU tract.
• Clinical infections are uncommon, but occur in immunocompromised and cancer patients.
• Commercial serogrouping kits are often used for identification.

Streptococcus pneumoniae

• Known as Pneumococcus.
• Characterized as lancet-shaped gram-positive diplococci.
• Demonstrates optimal growth in 5-10% CO2.
• Capsular polysaccharide increases its virulence, with 90 different serotypes.
• Alpha hemolytic

Hemolysis

• Colonies are mucoid or umbilicated (depressed center) because of autolytic enzymes.
• Most colonies survive for 48 hours.

Identification

• Sensitive to optochin disk
• Positive bile solubility
• Positive Quellung reaction.
• Commercial Kits for antigen detection are available to look for antigens in CSF, urine and serum

Optochin Susceptibility

• Sensitive to ethylhydrocupreine hydrochloride or P disk.
• P disk identify pneumococci.

Bile Solubility

• Bile salts speeds up natural autolysis.
• Sodium desoxycholate is used.
• Conducted via broth and plate methods.
• Positive result is S. pneumoniae confirmed.
• Identifies isolate as S. pneumoniae.
• Anti-pneumococcal serum and specimen are mixed; a positive reaction means the capsule swells.

S. pneumoniae (Clinical Significance)

• Virulence due to Capsular polysaccharide.
• Pneumonia (most common) causes rusty sputum.
• Can cause otitis media in young kids.
• Known to cause meningitis in adults.
• Commonly results in bacteremia
• Many strains are Resistant to penicillin.

Enterococcus

• E. faecalis or E. faecium most common.
• Normal flora of the oral region and GI tract.
• Belongs to Lancefield group D.
• Streptococcus bovis (viridance strep) group also group D.
• Exhibits alpha or gamma hemolysis

Enterococcus Virulence

• It can survive and grow in extreme conditions
• Has resistance to multiple antibiotics

Enterococcus: Clinical significance

• Causative agent in Nosocomial UTI, bacteremia,
• Also causes Endocarditis and wound infections

Enterococcus Identification

• Catalase negative.
• May exhibit pseudocatalase reaction.
• Bile-esculin positive
• Most of the time non-hemolytic on BAP
• Shows positive 6.5% salt growth
• PYR positive.
• Additional biochemical tests identify to species level.

Enterococci and Group D Streptococci

• Both are bile-esculin positive
• Differentiated by salt tolerance and PYR test.
• Enterococcus (positive) are salt tolerant
• Nonenterococcus Group D (negative) are not salt tolerant
• Group D is associated with colon cancer.
• Some Enterococcus: Negative Lancefield grouping

Bile-Esculin Agar

• Differentiates gram positive cocci, group D strep and Enterococcus.
• Contains 40% sodium desoxycholate which inhibits others that are not group D streptococci species.
• Contains esculin, ferric ammonium citrate.
• Esculin is hydrolyzes and changed to esculetin and glucose
• The reaction of esculetin and ferric ions results in black color on the agar
• Gram-positive bacteria bile-esculin positive enterococci results in a black precipitate on the culture.
• A negative Bile-Esculin test result yields no blackening of agar

6.5% Salt-Tolerance Test

• This differentiates gram positive cocci that will grow in 6.5% NaCl from those that are inhibited by a high concentration of salt.
• Inoculate, incubate, then observe for growth.
• A positive reaction demonstrates turbidity or color change (if media contains pH indicator).
• A negative reaction indicates growth of S. bovis group (a nonenterococcus).
• Bile-esculin positive, 6.5% salt is enterococci
• Bile-esculin positive, 6.5% salt negative is nonenterococcal group D streptococci

Viridans Streptococci

• Normal flora of the RT, GI, GU, skin.
• These are opportunistic pathogens which require higher CO2 levels.
• There are > 30 species with 5 groups: S. mitis group, S. mutans group, S. salivarius group, S. bovis group, and S. anginosus group.
• Lancefield grouping is non-groupable.
• S. anginosus group may be groups A, C, F, G, or N.
• S. bovis group have group D antigen.
• Enterococci also have group D antigen.
• Identified using "ruling out" process.
• Alpha or gamma streptococci considered viridans if not enterococci or pneumococci.
• Lack of beta-hemolysis differentiates from groups A, B, C, or G

Viridans

• This group results in greening and a-hemolytic.
• Includes ß-hemolytic and nonhemolytic species

Viridans Streptococci: Clinical Significance

• Most common cause of subacute bacterial endocarditis, especially in patients with damaged or artificial heart valves.
• Cause of Bacteremia - associated with hematologic cancers.
• S. gallolyticus (S. bovis group) is often associated with GI cancer.
• Can cause Transient, following dental procedures
• Cause of tooth decay and gingivitis (S. mutans)
• Cause of Wound infections and formation of brain abscesses

Streptococcus-like Bacteria (Bacteremia and Endocarditis)

• Includes Abiotrophia and Granulicatella, formerly known as nutritionally variant streptococci (NVS).
• Defective in vitamin B5 (pyridoxal) production.
• Can be suspected in positive blood culture if subculture fails to grow.
• Do not grow on BAP or CHOC.
• Demonstrate satellitism.

Satellite Phenomenon

• Occurs when a deficient organism is spread on BAP agar.
• A second organism (S. aureus) is single streaked.
• Tiny colonies are seen near the staph streak after incubation

Streptococcus-like Bacteria

• Includes Vancomycin-resistant Leuconostoc, which is PYR +, leucine aminopeptidase (LAP) +, and Bile Esculin -ve.
• Includes Pediococcus
• Gemella, another Streptococcus-like Bacteria which is PYR +, leucine aminopeptidase (LAP) +, and Bile Esculin -ve.
• Includes Lactococcus

Streptococcus-Like Bacteria Characteristics

• Resemble viridans.
• Present as NF in the mucous membranes and in the environment.
• Cause very rare infections such as endocarditis, bacteremia, and wound infections.
• Show alpha or gamma hemolysis.

Helpful Hints - Identification

• Hemolysis patterns should always be noted.
• Colony morphology: tend to be smaller and more translucent than staph colonies.
• A good Gram-stained smear (GPC) is helpful.
• Catalase test (negative) - helps to distinguish staph from strep.

Hemolysis

• Alpha hemolysis points to enterococci, group D, S. pneumoniae, or viridans strep.
• Test for enteroccoci, group D requires P disk as well as a bile solubility test
• Beta hemolysis often hints towards group A, B (but also C, F, G)
• Group A and B are detected through A disk, SXT disk, PYR, CAMP, or hippurate hydrolysis.
• Gamma hemolysis points to group B, enterococci, group D, or viridans
• A CAMP must be done utilizing hippurate hydrolysis as well as bile-esculin hydrolysis.