Streptococcus pyogenes Identification PDF

Catalase Negative Gram-Positive Cocci in Pairs and Chains Streptococcus Enterococcus Strep-like organisms Characteristics  GPC in chains or pairs  catalase negative  distinguishes streptococci from staphylococci  facultative anaerobes  some organisms capnophilic (­CO2)  gr...

Catalase Negative Gram-Positive Cocci in Pairs and Chains Streptococcus Enterococcus Strep-like organisms Characteristics  GPC in chains or pairs  catalase negative  distinguishes streptococci from staphylococci  facultative anaerobes  some organisms capnophilic (­CO2)  grow on BAP, CHOC, CNA, PEA  BAP is preferred for hemolysis demonstration Gram Stain Cell Wall Structure  typical GP cell wall (peptidoglycan and teichoic acid)  capsule (young cultures)  C (capsular) polysaccharide antigen Classification Schemes  same organism may have multiple names  four systems!!  hemolytic pattern  serologic grouping  common names  physiologic characteristics  Hemolytic Pattern  Exotoxins damage RBCs  Observe on BAP  Alpha: green, incomplete due to is the reduction of hemoglobin to methemoglobin in the medium surrounding the colony. This produces a green discoloration of the medium  Alpha prime: narrow alpha hemolysis with wide beta hemolysis. Or is a small zone of complete hemolysis surrounded by an area of partial lysis  Beta: clear, complete  Gamma: none Type of hemolysis on Sheep blood agar plate S. pneumoniae Streptococcus Non- or γ-hemolytic α-hemolytic group A and B Group D β- hemolytic Streptococcus 8 Serogrouping  Lancefield grouping  based on cell-wall antigens (C carbohydrate)  latex agglutination techniques (available commercially) used for grouping  not all streptococci can be identified through serogrouping  A, B, C, D, F, G Common Names  b strep or group A strep  Group B strep  Group D strep  a strep  Pneumococci  Viridance group  Enterococcus Physiologic Properties  based on biochemical reactions  each organism given a genus and species name S. Pyogenes (virulence)  Hemolysis caused by two toxins  Streptolysin S (SLS) oxygen stable  Streptolysin O (SLO) oxygen labile hemolysis may not be detected if incubated aerobically SLO protected from oxygen when BAP is stabbed or cut (localized anaerobic environment) also lyses WBCs and platelets Stimulates production of anti-streptolysin O (ASO) S. pyogenes  lipoteichoic acid and fibronectin binding protein : adhesion lipoteichoic acid Fibronectin binding protein fibronectin epithelial cells 13 S. pyogenes  M protein needed for virulence  Resist phagocytosis  Increases adherence to mucosal surfaces  Erythrogenic/pyogenic toxins Streptococcus toxic shock syndrome (STSS), like TSST-1  Fever, rash, organ damage  Streptokinase  Lyses blood clot (RBC, platelets, WBS)  Major pathogenesis factors – M protein Complement IMMUNE IgG r r r M protein peptidoglycan NON-IMMUNE fibrinogen r 15 r r S. pyogenes  Pharyngitis (strep thought)  scarlet fever  streptococcal toxic shock syndrome  skin or pyodermal infections  post-streptococcal sequelae  rheumatic fever  acute glomerulonephritis Pharyngitis/Tonsillitis  Strep throat  Common S. pyogenes disease  Seen most often in children  Rapid detection kits available (latex) Pharyngitis  Antigen Detection Tests  alternative to throat culture  fast  commercially available kits  high specificity but low sensitivity (60-95%)  sensitivity = probability of a positive test among patients with disease specificity = probability of a negative test among patients without disease  negative results should be confirmed by culturing Scarlet Fever and Streptococcal Toxic Shock Syndrome  Scarlet fever  Streptococcal pyogenic exotoxin  Fever with rash after bacterial infection  Streptococcal toxic shock syndrome (STSS)  Organ shut down and death  mainly due to erythrogenic/pyogenic toxins Pyodermal Infections  Impetigo  Erysipelas acute infection of the upper dermis and superficial lymphatics  Necrotizing Fasciitis – flesh eating bacteria Post-Streptococcal Sequelae  follow S. pyogenes infections  autoimmune in origin  acute rheumatic fever after pharyngitis  Fever and inflammation  Rheumatic heart disease  acute glomerulonephritis after pharyngitis or pyoderma  Streptococcus pyogenes (identification)  Group A strep, b Strep  not part of NF, although it can be asymptomatic in young children  Small (less than 1 mm), translucent colonies  b hemolytic  Small, pinpoint colony  Big zone of hemolysis  Hemolysis improved if BAP is stabbed, cut, or incubated anaerobically? Throat swab culture Purity plate (PP) Identification  Direct latex agglutination test  Culture : BAP- β-hemolytic  Serogrouping Lancefield group A  PYR (L-pyrrolidonyl arylamidase) positive  Bacitracin (A) disk susceptible  SXT (sulfamethoxazole –trimethoprim) disk resistant Serogrouping  commercial streptococcal serogrouping kits  latex agglutination From throat swab  Serological testing: look for microbial Ags Streptococcal Agglutination Test: Modern Rapid “Strep” Test Streptococcal antigenic extract Antibody Latex beads Serological testing: looks for Ag against the microbe: Modern Rapid “Strep” Test Sample: throat swab + ve - ve 27 Bacitracin (A) Disk Susceptibility  inhibited by bacitracin  test should only be performed on beta hemolytic streptococci  A disk used to ID group A streptococci  Group A strep is susceptible to the A disk  Group B strep is resistant to the A disk A Disk Resistant Susceptible Trimethoprim- Sulfamethoxazole (SXT)  two antibiotics  groups A and B are resistant  groups C, F, G are susceptible  Group C and G called S. disagalactia: cause disease similar to Group A. PYR Hydrolysis Test  test detects presence of enzyme PYRase  Hydrolyzes L-pyrrolidonyl-β-naphthylamide (PYR) to dimethylaminocinnamaldehyde  test is positive with Enterococcus , Group A and other organisms PYR Negative Positive Organisms possessing the enzyme L-pyrrolidonyl arylamidase can hydrolyze the disk substrate L-pyrrolidonyl-ß-naphthylamide to release L-pyrrolidone and ß-naphthylamide. Visual detection can be achieved by the addition of PYR/LAP Reagent or (DAMACA).the active ingredient, p- dimethylaminocinnamaldehyde, combines with the end-product ß-naphthylamide to form a red Schiff base. A positive reaction is a yellow to red color change  Typing 1. Traditional serotyping (M-T-R): - M protein - T (Lancefield group) - R protein 2. Current: - Sequencing of M protein gene 33 S. agalactiae  Group B Streptococci  NF of genitourinary tract (25% carrier)  polysaccharide capsule  Group B is for Baby  group B streptococci cause serious infection in Babies Hemolysis and Colonial Morphology  Grayish-white, flat, creamy colonies (> 1mm).  Most are beta hemolytic  larger colonies  very small zone of hemolysis Identification  Serogrouping Lancefield group B  Bacitracin (A) disk resistant  CAMP positive  Hippurate hydrolysis positive  Molecular analysis Antigen Detection Tests  commercial tests (latex agglutination)  detects antigen in  CSF  serum  urine  vaginal secretions  NF may cause false positive results in urine and vaginal specimens Disks  A disk resistant  SXT resistant CAMP Test  S. aureus produces b-lysin and group B streptococci produces CAMP factor  Enhanced hemolysis observed when CAMP factor and b-lysin act synergistically on BAP  Test organism streaked perpendicular to S. aureus, but not touching  Arrow-shaped hemolysis at junction of two streaks CAMP (A) Streptococcus (group B) shows a positive CAMP reaction. (B) Streptococcus pyogenes (group A) shows a negative reaction when inoculated at a right angle to (C) Staphylococcus aureus Hippurate Hydrolysis  detects the enzyme hippuricase  hydrolyzes sodium hippurate (N- benzoylglycine) into sodium benzoate and glycine  ninhydrin reacts with glycine to form a purple color Hippuric acid  2-hour rapid test  other organisms are positive too! Hippurate Positive = purple Negative = no color Positive Negative Molecular Detection  Beta strep screen of pregnant women  Group B strep screen using real time PCR  98% recovery for PCR  compared with 75% for culture Clinical Significance  neonatal sepsis and meningitis  both due to organism being vaginal NF  infants become colonized during birth  few develop disease  UTI in adults  skin and soft tissue infections  postpartum (after birth) fever  sepsis β-Hemolytic Streptococci Groups C, F and G  NF of skin, nasopharynx, GI tract, GU tract  Clinical infections are uncommon  Immunocompromised  Cancer patients  Commercial serogrouping kits  Usual ID method Streptococcus pneumoniae  Pneumococcus  lancet shaped GP diplococci  optimal growth in 5-10% CO2  capsular polysaccharide increases virulence (90 different serotypes) Streptococcus pneumoniae Hemolysis  alpha hemolytic  colonies  mucoid (polysaccharide capsule) or,  umbilicated (depressed center) caused by autolytic enzymes (most colonies lived only for 48 hours) Mucoid Colonies Umbilicated Colonies Identification  Optochin disk sensitive  Bile solubility positive  Quellung reaction positive  Commercial kits for antigen detection available  CSF, urine, serum Optochin Susceptibility P Disk  sensitive to ethylhydrocupreine hydrochloride or P disk  Remember!  P disk identify pneumococci P Disk Bile Solubility  bile salts accelerates natural autolysis in penumococci  sodium desoxycholate  broth and plate methods  positive bile solubility = S. pneumoniae Bile Solubility  Place 1 – 2 drops of bile salts on colony  Incubate  Observe for lysis Quellung Reaction  Identifies isolate as S. pneumoniae  Anti-pneumococcal serum mixed with specimen or isolate ==> capsule becomes more refractile and swollen S. pneunoniae  Virulence  Capsular polysaccharide  Clinical significance  pneumonia (most common)  pneumococcal pneumonia ==> rusty sputum  otitis media – ear infections in young kids  meningitis (most common cause in adults)  bacteremia Read about antibiotic sensitivity. Many strains are Resistant to penicillin Enterococcus  E. faecalis or E. faecium most common  NF of oral and GI tract  Lancefield group D  Streptococcus bovis (viridance strep) group also group D  Alpha or gamma hemolysis Enterococcus  Virulence  Growth in extreme conditions  Resistance to multiple antibiotics  Clinical significance  Nosocomial UTI, bacteremia  Endocarditis  Wound infections Enterococcus Identification  Catalase negative  careful: can exhibit pseudocatalase reaction  Bile-esculin positive  Most of the time non-hemolytic on BAP  6.5% salt growth  PYR positive  Additional biochemical tests identify to species level Enterococci and Group D Streptococci  both are bile-esculin positive  salt tolerance and PYR test to distinguish Enterococcus (positive) from nonenterococcus Group D (negative)  Group D is associated with colon cancer  Some Enterococcus: Negative Lancefield grouping Bile-Esculin Agar  Differentiate group D strep and Enterococcus from other gram positive cocci  Contains 40% sodium desoxycholate  bile salt inhibits most non-group D streptococci  Contains esculin, ferric ammonium citrate  esculin hydrolyzed ® esculetin + glucose  esculetin + ferric ions ® black color Bile-Esculin Agar  Positive result = blackening of agar  Negative result = no blackening of agar 6.5% Salt-Tolerance Test  Differentiate gram positive cocci that will grow in 6.5% NaCl from those that are inhibited by high concentration of salt  inoculate, incubate, observe for growth  Positive = turbidity or color change if media contains pH indicator  Positive = Enterococcus  Negative = S. bovis group (nonenterococcus) 6.5% Salt-Tolerance Test Positive = cloudy Negative = clear Change in pH = yellow No change in color Clear Turbid Conclusions  bile-esculin positive, 6.5% salt-positive GPC ® enterococci  bile-esculin-positive, 6.5% salt negative GPC ® nonenterococcal group D streptococci Viridans Streptococci  NF of RT, GI, GU, skin  Opportunistic pathogens  May require ­ CO2  > 30 species  5 groups  S. mitis group  S. mutans group  S. salivarius group  S. bovis group  S. anginosus group Viridans Streptococci  Lancefield grouping (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  Identified by “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 Streptococci  Viridans = greening, α-hemolytic  β-hemolytic and nonhemolytic species Clinical Significance  Most common cause of subacute bacterial endocarditis  patients with damaged or artificial heart valves  Bacteremia  Associated with hematologic cancers  S. gallolyticus (S. bovis group) associated with GI cancer  Transient, following dental procedures  Tooth decay, gingivitis (S. mutans)  Wound infections, brain abscesses Streptococcus-like Bacteria (Bacteremia and Endocarditis)  Abiotrophia and Granulicatella  Formerly known as nutritionally variant streptococci (NVS)  Defective in vitamin B5 (pyridoxal) production  Suspect in positive blood culture if subculture fails to grow  Do not grow on BAP or CHOC  Demonstrate satellitism Satellite Phenomenon  deficient organism spread on BAP agar  second organism (S. aureus) single streaked  after incubation, tiny colonies seen near staph streak Streptococcus-like Bacteria  Vancomycin-resistant  Leuconostoc PYR +, leucine aminopeptidase (LAP), and Bile Esculin –ve.  Pediococcus  Other  Gemella: PYR +, leucine aminopeptidase (LAP) +, and Bile Esculin –ve.  Lactococcus Streptococcus-Like Bacteria  resemble viridans  NF of mucous membranes and in environment  very rare infections  endocarditis  bacteremia  wound infections  alpha or gamma hemolysis Helpful Hints - Identification  Hemolysis  Colony morphology  smaller colonies than staph  more translucent than staph colonies  Good gram-stained smear (GPC)  Catalase (negative) - distinguish staph from strep Hemolysis  Alpha ® enterococci, group D, S. pneumoniae, viridans strep  P disk (or bile solubility), bile-esculin hydrolysis, 6.5% salt, PYR  Beta ® group A, B (also C, F, G)  A disk, SXT disk, PYR, CAMP (or hippurate hydrolysis), serogrouping  Gamma ® group B, enterococci, group D, viridans  CAMP (or hippurate hydrolysis), bile-esculin hydrolysis, 6.5% salt, PYR GPC, Catalase Negative alpha hemolysis optichin (P) disk susceptible resistant S. pneumoniae bile esculin positive negative PYR viridans 6.5% NaCl positive negative enterococcus group D GPC, Catalase Negative Beta Hemolysis A Disk suscpetible resistant S. pyogenes CAMP or PYR + Hippurate positive negative S. agalactiae bile esculin positive negative enterococcus group D not A, B, or D PYR & NaCl + PYR & NaCl -ve viridans GPC, Catalase Negative gamma hemolysis CAMP (hippurate) positive negative S. agalactiae bile esculin positive negative 6.5% salt viridans PYR positive negative enterococci group D