Streptococcaceae Chapter 15: Streptococcus, Enterococcus, and Other Catalase-Negative Gram-Positive Cocci

Streptococcaceae: Genera Streptococcus – major cause of human infection Enterococcus – frequent cause of human infection Aerococcus – occasionally endocarditis, meningitis, urinary tract infection Lactococcus – rare human pathogen Pediococcus – NF of lower GI tract, occasional isolate from abscess Leuconostoc – infrequently isolated from blood, wound, abscess Gemella – rare isolate of upper RT infection

Streptococcaceae: Classification Medical importance: Streptococcus, Enterococcus Differentiation on CBA – partial, complete, nonhemolytic (, , or none) Serologic classification developed 1930’s by Lancefield CHO (C substance) extracted from cell wall Group A through U Some Strep don’t have this antigen, not ID by this method Lancefield typing used today primarily for beta hemolytic Group A, B

Classification Physiologic or biochemical characteristics used to differentiate species within Streptococci and Enterococci In future, DNA probes and hybridization, PCR DNA amplification tests will be used (greater sensitivity and specificity)

Gram Stain Gram (+) cocci, in pairs or chains (most often seen in broth culture); cell division occurs in 1 plane Strep pneumoniae is lancet shaped diplococcus Strep quickly lose ability to retain crystal violet in Gram stain; appear G (-) in old cultures

Strep Characteristics Form capsule More fastidious than Staphylococci, require enriched media such as CBA Hemolytic on CBA and source of blood (sheep vs. rabbits) may affect hemolysis, clinical labs use 5% sheep RBCs in CBA Group A Strep two hemolysins: Streptolysin O - oxygen labile; stabbing CBA after inoculation provides lower oxygen tension to identify strains that only produce streptolysin O Streptolysin S – oxygen stable

Characteristics Addition of sulfamethoxazole & trimethoprim (SXT) to CBA may be used to suppress growth of NF and allow better recovery of beta hemolytic Strep Microaerophilic, good growth require reduced oxygen (candle jar or CO2 incubator) Nutritionally deficient Strep may require vitamin B6 or pyridoxal for growth and may be found satelliting Staph aureus Strep pneumoniae is sensitive to cold and may fail to grow if refrigerated

Strep Differentiation From Other Genera Other Gram(+) cocci: Strep = catalase(-) Staph & Micrococci are catalase(+) Other cocci: Neisseria are Gram(-) May be confused with Strep that lose G(+) as they age However, Neisseria = oxidase(+)

Differentiation Within Strep Genus Respiratory Tract by CBA hemolysis: Viridans group (Strep mitis, Strep salivarius, Strep sanguis) - partial (α) hemolysis, NF Strep pneumoniae – partial (α) hemolysis Strep pyogenes – complete (β) hemolysis, may be pathogen Differential diagnosis by clinical syndrome: Group A – sore throat Group B – neonatal meningitis

Strep pyogenes (Group A) Sensitive to Bacitracin (0.04 units, Taxo A disk) Resistant to SXT disk (both Gp. A & B) PYR hydrolysis: Production of pyroglutamyl aminopeptidase; both Gp. A & Enterococcus = (+)

Strep agalactae (Group B) CAMP (Christie, Atkins, Munch-Peterson) test – extracellular protein that enhances Staph aureus hemolysin Hippurate hydrolysis (hippuricase) – hippurc acid to Na benzoate + glycine (amino acid) Growth in 6.5% NaCl (Gp. B & Enterococcus) Resistance to SXT disk (both Gp. A & B)

Group D: Enterococcus, Streptococcus bovis CBA - may be partial, complete, nonhemolytic (α, β, none) Bile esculin agar - all Group D grow in bile, hydrolyze esculin = growth + blackening on agar 6.5% NaCl – Enterococcuss = (+) growth PYR hydrolysis – Enterococcus = (+)

S. pneumo & Viridans Group CBA - both partial (α) hemolysis Taxo P disk- Optochin (ethyl hydrocupreine hydrochloride) susceptibility, detergent that activates autolytic enzyme, lyse cell wall of Strep pneumoniae Bile solubility – bile salt (Na desoxycholate) activate autolytic enzyme of Strep pneumoniae (test - 37°, 30 min.) dissolve isolated colonies Most labs do not speciate the Viridans group (Strep mitis, Strep salivarius, Strep sanguis) except for blood isolates or epidemiology

Serology Tests Serogroups – Lancefield typing Group A-H, K-V Group A Serotypes - >80; based on M protein of cell wall Quellung test - for Strep pneumoniae 84 serotypes based on specific soluble substance in capsule specific antisera binds to substance, under microscope capsule appears swollen

Strep pyogenes Virulence: Extracellular products Streptolysin S – alters membrane permeability; lyses RBCs, leukocytes, and other cells containing sterols in membrane Streptolysin O – forms membrane penetrating channels (porin) leading to membrane defects and cell lysis Cytotoxic to RBCs and many tissue cells, particularly cardiotoxic Antigenic - for strep throat infections (but not skin), anti-streptolysin O (ASO) antibody used to diagnose recent infection

Strep pyogenes Virulence: Extracellular products Erythrogenic toxin Act as superantigen - fever, cardiotoxic, enhanced susceptibility to endotoxin shock, suppress antibody response Rash seen in scarlet fever; due to delayed hypersensitivity reaction to other streptococcal antigens enhanced by toxin Toxin involved in toxic shock syndrome similar to Staph. aureus

Strep pyogenes Virulence: Extracellular products Streptokinase – cleaves plasminogen to plasmin, then acts as protease to lyse fibrin clots Hyaluronidase – spreading factor DNAse – degrades accumulated inflammatory exudate DNA from leukocyte disintegration; antigenic and antibodies used to diagnose recent Strep infection (including skin infections)

Strep pyogenes Virulence: Cell Associated Factors M protein Fibrous protein on cell surface MAJOR virulence factor for group A Strep >80 different types Protects MO from opsonization and phagocytosis by binding to fibrinogen Also play role in adherence Some types of M protein act as super antigen causing shock in patient Poorly immunogenic; antibodies, if made, are protective, long lasting, type specific

Strep pyogenes Virulence: Cell Associated Factors Lipoteichoic acid mediates attachment to epithelial cells Capsule hyaluronic acid (note organism also produces a hyaluronidase) antibodies to capsule are not protective against infection

Strep agalactiae Virulence: Extracellular Products Hemolysin - increase vascular permeability in lungs, leading to respiratory distress Neuraminidase – cleaves sialic acid residues, involved in hyaline membrane disease of lung Hyaluronidase – spreading factor DNAse – spreading factor Protease – spreading factor

Strep agalactiae Virulence: Cell associated Factors Capsule – antibodies are type specific and protective Lipoteichoic acid – for attachment

Strep pneumoniae Virulence: Extracellular Products Pneumolysin O cytolytic for RBCs and other tissue cells Cytoplasmic rather than extracellular protein, released when bacteria lyse Acts as porin to disrupt host cell membranes Leukocidan Neuraminidase Hyaluronidase IgA protease

Strep pneumoniae Virulence: Cell Surface Structures Capsule – most important virulence factor > 80 different types capsular polysaccharide Antibodies against capsule type specific and protective (used for vaccine)

Streptococcus Clinical Significance A wide variety of diseases: Suppurative - pus producing lesions Toxemia - Clinical symptoms suggesting toxin Infants - more susceptible, with prolonged, low grade infections; rarely nephritis and rheumatic fever follow Older children and adults - more acute and self-limited infections; occasional complication of nephritis and rheumatic fever (with no pus or MO present)

Streptococcus: Clinical Significance Complete (ß) hemolytic: Gp. A, B, and C most virulent Gp. A greatest number infections Partial (α) hemolytic & non hemolytic: Viridans group found as NF in RT and GI tract, most common cause of subacute bacterial endocarditis Step pneumoniae leading cause of death due to bacterial infection throughout the world, particularly in elderly and very young

Strep pyogenes Infections: Pyoderma (Impetigo) Superficial skin infection characterized by vesicular rash, becomes pustular, in later stages thick crust Staph aureus may be secondary invader Endemic in tropical areas caused by M serotypes different from those that cause pharyngitis Spread by close, direct contact Disease starts with skin colonization, followed by invasion through abrasions Type specific antibodies to M are made, but antibodies to Streptolysin O are uncommon Most serious complication is acute glomerulonephritis, occur 2-3 weeks post infection

Strep pyogenes Infections: Erysipelas Diffuse, erythematous skin infection Most often on face, occurs following pharyngitis

Strep pyogenes Infections Wound infections - occasionally following trauma, due to simple contamination of wound Cellulitis – subcutaneous tissue, accompanied by lymphangitis and abscess; typically exhibit chills, fever, marked signs of toxicity Necrotizing fasciitis (”flesh eating bacteria”) – fibrous tissue deep in skin; toxin destruction of sheath that covers muscle

Strep pyogenes Infections: Myositis Invasion of muscle tissue Extensive muscle necrosis, overwhelming sepsis Usually fatal

Strep pyogenes Infections: Pharyngitis (Strep Sore Throat) Most common disease caused by Strep pyogenes Spread by droplets of respiratory secretions or via food or water Abrupt onset of sore throat, malaise, headache, fever Throat erythematous with grayish-white exudate Tonsils and lymph nodes are enlarged; tonsils may become infected chronically May spread to sinuses and middle ear Usually self-limited, penicillin used to prevent secondary complications such as rheumatic fever

Strep pyogenes Infections: Scarlet Fever From URT infection Strep lysogenized by phage with gene for erythrogenic toxin Symptoms similar to pharyngitis - erythematous rash upper chest, spreads to rest of body, ~1 week; followed by weeks of extensive desquamation of skin “strawberry tongue” - red spots on yellowish-white tongue “raspberry tongue” - followed by peeling, red beefy tongue Immunity to toxin is demonstrated by Dick test: Inject toxin in skin, produces erythema in absence of antibody If no erythema, indicates antibodies present, patient infected Erythrogenic toxin may also be involved in causing a toxic-shock like syndrome

Non-suppurative Complications Strep pyogenes: Rheumatic Fever Inflammatory disease; involve heart, joints, subcutaneous tissue, CNS Primarily in children 6-15 yrs. 1-5 weeks after Strep throat Major manifestations - polyarthritis, carditis, chorea, erythema marginatum Carditis can involve all layers of heart, can lead to rheumatic heart disease - chronic, progressive damage to heart, possibly death

Rheumatic Fever By autoimmune, cross-reacting antibodies made against Strep that react with self tissues Attracts host reactive cells, Complement gets activated and leads to cellular damage Diagnosis by serum antibody test - ASO assay Prevent by treating Strep throat infection with penicillin

Non-suppurative Complications Strep pyogenes: Acute Glomerulonephritis Occur 2-3 weeks following pharyngitis or pyoderma Edema, hypertension, headache, malaise, circulatory congestion, hematuria Only associated with specific nephritogenic M serotypes of Gp. A Strep Immune complex disease - deposition of circulating Strep antigens and antibodies complexes in glomeruli of kidney Damage due to host reactive cells and subsequent Complement activation

Strep agalactiae Infections NF of adult vagina, urethra, rectum, occasionally pharynx Sexually transmitted disease (STD) Adults - rare infections; URT, meningitis, bacteremia, endocarditis Newborn - most serious disease cases Use to be major cause of puerperal fever (postpartum sepsis, child-bed fever)

Strep agalactiae Infection: Newborn Early onset disease Infected in utero or at birth MO gain access via RT, symptoms of respiratory disease Septicemia, meningitis may occur High mortality rate Late onset disease 7-10 days after birth, MO rarely found in mother prior to birth MO by direct contact with newborn mucosal surfaces Meningitis or osteomyelitis, possibly bacteremia Mortality rate high, but lower than early-onset disease

Streptococcal Group C, D Infections Group C – similar to Gp. A, but without non-suppurative sequelae complications Group D – NF of GI tract Common cause of UT infections, subacute bacterial endocarditis, abdominal abscesses, wound infections Strep bovis in blood suggest surface of colon compromised; associated with carcinoma of colon; proposed as early screen for colon cancer

Viridans Group Infections NF of RT, GI tract, genital tract Most common cause of subacute bacterial endocarditis, colonize damaged heart valves Strep mutans plays a role in dental decay

Strep pneumoniae Infections Found as NF in URT in 15% of children and 5% of adults; carrier state is sporadic Probable pathogen when most predominant MO isolated Leading cause of bacterial pneumonia, particularly in very young, elderly, those with immunological deficiencies Mortality rate is 5-7%. Bacteremia is common, meningitis may occur

Strep pneumoniae Infections Meningitis – not as common as other causes of meningitis except in children under 1 year of age where it is second most common cause of meningitis; mortality rate is high ~40% Otitis media – most common cause of middle ear infection in children (1/3 of cases) Also cause purulent sinusitis and peritonitis

Antimicrobial Susceptibility Penicillin for groups A, B, C; erythromycin for individuals allergic to penicillin. Susceptibility testing is not necessary. Enterococcus and other group D – must do sensitivity testing; now strains of Enterococcus faecalis resistant to all antibiotics Viridans group Strep – do sensitivity testing Strep pneumoniae – use penicillin, but now resistant strains, so sensitivity testing is necessary

Pneumococcal Vaccine Use of purified polysaccharide Children: heptavalent pneumococcal vaccine (PCV7) – vaccine of 7 serotypes S. pneumoniae Adults: 23 capsular polysaccharides (PS23) - vaccine for 23 strains most likely to cause pneumonia Given to most likely infected - young, old, chronic respiratory problems

Class Assignment Textbook Reading: Chapter 15 Streptococcus, Enterococcus, and Other Catalase-Negative Gram-Positive Cocci Key Terms Learning Assessment Questions

Case Study 2 - Streptococcus A 62-year-old man with a history of chronic obstructive pulmonary disease (COPD) came to the emergency department because of a fever of 40°C, chills, nausea, vomiting, and hypotension. The patient also produced tenacious, yellowish sputum that had increased in quantity over the preceding 3 days. His respiratory rate was 18 breaths/min, and his blood pressure was 94/52 mmHg.

Case Study 2 Chest radiographic examination showed extensive infiltrates in the left lower lung that involved both the lower lobe and the lingula. Multiple blood cultures and culture of the sputum yielded S. pneumoniae. The isolate was susceptible to cefazolin, vancomycin, and erythromycin but resistant to penicillin.

Case Study 2 - Questions 1. What predisposing condition made this patient more susceptible to pneumonia and bacteremia caused by S. pneumoniae? What other populations of patients are susceptible to these infections? What other infections does this organism cause, and what populations are most susceptible? 2. What infections are caused by S. pyogenes, S. agalactiae, and viridans streptococci?

Case Study 2 –Questions 3. S. pyogenes can cause streptococcal toxic shock syndrome. How does this disease differ from the disease produced by staphylococci? 4. What two non-suppurative diseases can develop after localized S. pyogenes disease?