MLAB 2434: Microbiology Keri Brophy-Martinez Streptococci, Enterococci and Other Catalase-Negative Gram Positive Cocci

Streptococcus and Enterococcus: General Characteristics Members of the Streptococcaceae family Facultatively anaerobic Aerotolerant Catalase negative

Streptococcus and Enterococcus: General Characteristics Most are typically spherical; some may appear elongated or ovoid They may appear in chains or pairs

Streptococcus and Enterococcus: Habitat and Clinical Infections Normal Flora Respiratory tract Gastrointestinal tract Urogenital tracts Clinical Infections Upper and lower respiratory tract infections Urinary tract infections Wound infections Endocarditis

Streptococcus and Enterococcus: Cell Wall Structure Thick peptidoglycan layer Teichoic acid Carbohydrate layer present Used in Lancefield grouping of Streptococcus spp. Capsule Virulence factor S. pneumoniae

Classification Overview Physiologic characteristics Pyogenic: produce pus Lactococci: found in dairy products Enterococci: normal gut flora Viridans: normal URT flora Hemolysis J. H Brown Alpha, beta, gamma classifications Serological grouping Typing of C carbohydrate Lancefield group Performed only on β-hemolytic hemolysis Biochemical Based on reaction of isolate

Classification: Hemolysis J.H. Brown- 1903 Grouped streps on ability to lyse RBCS Alpha Beta Gamma Alpha-prime

Hemolysis Patterns Alpha (α): Greenish discoloration Caused by partial lysis of RBCs in media

Hemolysis Patterns Beta (ß): Complete lysis of RBCs Produces a clear, colorless zone

Hemolysis Patterns Gamma : Colonies show no hemolysis or discoloration Called non-hemolytic

Classification: Serological Grouping Rebecca Lancefield – 1930 Based on presence of carbohydrates in cell wall Groups A, B, C, and D most significant Typing done on beta-hemolytic colonies

Classification: Biochemical Identification/Susceptibility Bacitracin “A” disk or “Taxo A” disk 0.04 units Identifies Group A streptococci (S. pyogenes) Zone of inhibition is presumptive ID of Grp. A strep Group A streptococcus is susceptible to “A” disk (left)

Biochemical Identification/Susceptibility Optochin P disk or“Taxo P” disk Differentiates S. pneumoniae from other alpha-hemolytic streptococci

Biochemical Identification Bile solubility test Detects amidase enzyme Under bile salt or detergent lyses cell wall Clearing through lysis of colonies Diagnostic for S. pneumoniae

Biochemical Identification PYR hydrolysis Substrate L-pyrrolidonyl-b- napthlyamide (PYR) is hydrolyzed by the enzyme pyrrolidonyl arlamide Group A Streptococci and Enterococcus sp. posses the necessary enzyme. More specific than Bacitracin for Group A streptococci The disk on the right has turned a red color, indicating a indicating a positive reaction. The left disk remains a yellow color indicating a negative result.

Biochemical Identification Hippurate hydrolysis Differentiates Group B streptococci from other beta hemolytic streptococci Group B streptococci hydrolyzes sodium hippurate forming a purple color

Biochemical Identification CAMP test Christie,Atkins, Munch- Petersen Detects the production of enhanced hemolysis that occurs when b-lysin and the hemolysins of Group B streptococci come in contact with each other Group B streptococci showing the classical “arrow-shaped hemolysis near the staphylococcus streak

Biochemical Identification Bile esculin hydrolysis Ability to grow in bile and hydrolyze Esculin Characteristic of streptococci that possess group D antigen and Enterococci Both Group D streptococci and enterococci produce a positive (top) bile Esculin hydrolysis test.

Biochemical Identification Salt Tolerance Growth in 6.5% NaCl broth Differentiates Group D streptococci from enterococci Enterococcus= POSITIVE Tube on left Group D Streptococcus= NEGATIVE Tube on right

Non-culture Identification Slide agglutination kits Latex beads are coated with group specific anti-serum, which clump when mixed with a small amount of colony from the specific Streptococcus sp. Nucleic Acid Probes Detect genes for specific groups

Slide Agglutination Tests

Slide Agglutination Tests

Break Time!

Virulence Factors: Streptococcus pyogenes Fimbrae: Protein F Attachment and adherence M protein: Resistance to phagocytosis Hyaluronic acid capsule: Prevents phagocytosis Lipoteichoic acid: Adheres to molecules on host epithelial cells

Virulence Factors: Streptococcus pyogenes Hemolysins Streptolysin O (O2 labile) detected in ASO titers Streptolysin S (O2 stable) Causes hemolysis on plates Erythrogenic toxin/Streptococcal pyogenic exotoxin: Scarlet fever Enzymes Streptokinase DNases Hyaluronidase – “spreading factor”

Clinical Conditions: Streptococcus pyogenes(Group A)

Clinical Conditions: Streptococcus pyogenes(Group A) Pyodermal infections Impetigo: weeping lesion Erysipelas Cellulitis Wound Infections Erysipelas due to Streptococcus pyogenes

Clinical Conditions: Streptococcus pyogenes(Group A) Scarlet Fever Starts with pharyngitis and causes rash on trunk and extremities Due to untreated Group A infections

Invasive Group A Streptococcal Infections Streptococcal toxic shock syndrome Multi-organ system failure similar to staphylococcal toxic shock Initial infection may have been pharyngitis, cellulitis, peritonitis, or other wound infections

Invasive Group A Streptococcal Infections Cellulitis/Necrotizing Fasciitis Severe form of infection that is life-threatening Bacteremia and sepsis may occur In patients necrotizing fasciitis, edema, erythema, and pain in the affected area may develop Streptococcal myositis resembles clostridial gangrene

Post–Group-A Streptococcal Infections Rheumatic fever Fever Inflammation of the heart, joints, blood vessels, and subcutaneous tissues Chronic, progressive damage to the heart valves (most evidence favors cross-reactivity between Strep. antigens and heart tissue) ASO titer will be elevated

Post–Group-A Streptococcal Infections Acute glomerulonephritis (AGN) Follows either cutaneous or pharyngeal infections More common in children than adults Antigen-antibody complexes deposit in the glomerulus Inflammatory response causes damage to the glomerulus and impairs the kidneys

Laboratory Diagnosis: Group A Streptococcus Grams stained wound smear showing gram-positive cocci in chains with numerous “polys” (PMNs)

Laboratory Diagnosis: Group A Streptococcus Colony morphology Transparent, smooth, and well-defined zone of complete or b- hemolysis

Laboratory Diagnosis: Group A Streptococcus Identification Catalase-negative Bacitracin-susceptible PYR-positive Hippurate hydrolysis- negative Slide agglutination Group A streptococci is susceptible to Bacitracin disk (left); The right shows resistance

Group B b-Hemolytic Streptococcus (Streptococcus agalactiae) Colonize the urogenital tract of pregnant women (10-30% rate – can cause OB complications such as premature rupture of membranes and premature delivery) Mother fails to pass protective antibodies to fetus Cause invasive diseases in newborns Early-onset infection Late-onset disease

Invasive Disease in the Newborn Early Onset Late-Onset Age of Onset < 7 days 7 – 30 days Median age of onset 1 hour 27 days Maternal complications of labor Common Less common Incidence of prematurity 25% Source of Organism Maternal genital tract Maternal genital tract; nosocomial; community Clinical presentation Nonspecific (35-55 %) Meningitis 5-10 % Respiratory diseases 35-55 % Focal Meningitis 25-35 % Types I, II III, V III (75%) Mortality Rate 5-15 % 2-10 %

Invasive Streptococcus agalactiae Infections In adults Occurs in immunosuppressed patients or those with underlying diseases Often found in a previously healthy adult who just experienced childbirth

Laboratory Diagnosis: Streptococcus agalactiae Colony morphology Small Grayish-white Mucoid, creamy Narrow zone of b-hemolysis

Laboratory Diagnosis: Streptococcus agalactiae Presumptive Identification tests Gram stain- GPC in chains Catalase-negative Bacitracin-resistant Bile esculin- negative Does not grow well in 6.5% NaCl. CAMP- positive Slide agglutination S. agalactiae shows the arrow-shaped hemolysis near the staphylococcus streak, showing a positive test for CAMP factor

Streptococcus pneumoniae General characteristics Inhabits the nasopharyngeal areas of healthy individuals Typical opportunist Possess C substance Virulence factors Polysaccharide capsule

Clinical Conditions: Streptococcus pneumoniae Most common cause of bacterial pneumonia Meningitis Bacteremia Sinusitis/otitis media Most common cause of otitis media in children < 3 years

Laboratory Diagnosis: Streptococcus pneumoniae Microscopic morphology Gram-positive cocci in pairs; lancet-shaped (somewhat oval in shape)

Laboratory Diagnosis: Streptococcus pneumoniae Colony morphology Smooth, glistening, wet-looking, mucoid a-Hemolytic CO2enhances growth As colony ages, autolytic collapse causes “checker shape”

Laboratory Diagnosis: Streptococcus pneumoniae Identification Catalase negative Optochin-susceptibility- test–susceptible Bile-solubility-test– positive

Identification Schema

Enterococcus Species Clinically Significant Isolates E. faecalis E. faecium Opportunistic pathogens In the GI tract, genitourinary tract and oral cavity Associated infections Bacteremia Urinary tract infections Wound infections Endocarditis Hospital-acquired Infections

Laboratory Diagnosis: Enterococcus Species Microscopic morphology Cells tend to elongate Colony morphology Small, grey Most are non-hemolytic, although some may show a- or, rarely, b-hemolysis Possess Group D antigen

Laboratory Diagnosis: Enterococcus Species Identification tests Catalase: may produce a weak catalase reaction Hydrolyze bile esculin Differentiate Group D from Enterococcus sp. with 6.5% NaCl or PYR test Important to identify Enterococcus from non-Enterococcus, because Enterococcus must be treated more aggressively.

Identification Schema Or PYR disk

Other Streptococcal Species Viridans group (Viridans means “green”) Members of the normal oral, nasopharyngeal flora, GI tract and female genital tract Most are  hemolytic but also includes nonhemolytic species The most common cause of subacute bacterial endocarditis (SBE) Also involved with gingivitis and dental carries PYR= negative Optochin= negative Bile solubility= negative

Viridans 5 groups Anginosus S. anginosus, S. intermedius, S. constellatus Mitis S. sanguig, S. parasanguis, S. gordonii, S. crista, S. infantis, S. mitis, S. oralis, S. oralis, S. peroris Mutans S. criceti, S. downei, S. macacae, S. mutans, S. rattus, S. sobrinus Salivarius S. salivarius, S. thermophilus, S. vestibularis Bovis S. equinus, S. gallolyticus,S. infantarius, S. alactolyticus

Abiotrophia & Granulicatella Once referred to as Nutritionally variant streptococci (NVS) Causes endocarditis and otitis media Normal flora of oral cavity Requires pyridoxal to grow (can satellite around Staph, E. coli, Klebsiella, Enterobacter and yeasts)

Streptococcus and Enterococcus

Streptococcus and Enterococcus Treatment Generally, streps are not routinely tested for susceptibility since penicillin drug of choice. If the patient is allergic to pen use erythromycin. Antibiotic resistance seen with Enterococcus, use vancomycin

References http://archive.microbelibrary.org/ASMOnly/Deta ils.asp?ID=2566 http://www.goodtoknow.co.uk/health/Scarlet- fever http://onwardstate.com/2009/12/10/keep-your- goals-to-yourself/ Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.