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GRAM POSITIVE COCCI

STREPTOCOCCI

STREPTOCOCCI
Gram (+) spherical cells, arranged in chains or pairs Grayish pinpoint translucent to slightly opaque colonies Catalase (-), oxidase (-) Growth is enhanced by blood, serum or glucose incorporated in

STREPTOCOCCI
Behave-like facultative anaerobe, some species require increased CO2 for growth Aerotolerant anaerobe (peptostreptococci obligate anaerobe

STREPTOCOCCI
ALL streptococci EXCEPT the viridans group have a layer of C carbohydrate serological classification Young cultures are characterized by the presence of capsule Are commonly found as part of normal human flora.

STREPTOCOCCI
However, when these organism gain access to normally sterile sites they can cause life threatening infection. S. pyogenes and S. pneumoniae - notorious pathogens Aerococcus, Lactobacillus, Leuconostoc and Pediococcus resemble streptococci

STREPTOCOCCI
Colony variation a.Mucoid b.Matte or rough large amount of CHON c.Smooth/glossy little amount of CHON

A. ACADEMIC/BERGEYS CLASSIFICATION BASED ON TEMPERATURE

CLASSIFICATION OF STREPTOCOCCI

1. PYOGENIC GROUP
Will not grow neither on 10C or 45C Produce pus; mostly -hemolytic Species: Streptococcus pyogenes

2. VIRIDANS GROUP
Will grow at 45C NOT part of the lancefield group Alpha hemolytic OR Non hemolytic Species: S. salivarius, S. mutans, S. mitis, S. sanguis

3. ENTEROCOCCUS
Will grow both at 10C and 45C Normal flora of human intestine Species: E. faecalis

4. LACTIC GROUP
Will grow on 10C Non hemolytic organism Often found in dairy products Species: S. lactis

B. SMITH AND BROWNS CLASSIFICATION BASED ON HEMOLYTIC PATTERN

CLASSIFICATION OF STREPTOCOCCI

1. ALPHA-HEMOLYTIC STREPTOCOCCI
Partial/incom plete hemolysis of RBC around colony Green discoloration around colony Species: S.

2. BETA-HEMOLYTIC STREPTOCOCCI
Complete hemolysis of RBCs around the colony Clear area/zone around colony Yellow discoloration Species: S. pyogenes, S. agalactiae

3. GAMMA-HEMOLYTIC STREPTOCOCCI
NO lysis of RBCs around colony/red cells immediately surrounding the colony are unaffected (no change) Species: E. faecalis (GROUP D)

C. LANCEFIELD BASED ON THE ANTIGENIC NATURE OF CELL WALL CARBOHYDRATES C POLYSACCHARIDE Rebecca Lancefield found out that C carbohydrate can be extracted from the streptococcal cell wall by placing the organism in dilute acid and heating for 10 min.

CLASSIFICATION OF STREPTOCOCCI

GROUP A STREPTOCOCCI
Pathogenic to man Not considered part of the normal flora Acquired thru contaminated droplets by cough or sneeze Species: S. pyogenes fever producing bacteria, flesh eating bacteria (involves deeper tissues and organs)

GROUP A STREPTOCOCCI
Principal virulence factor: Mprotein (type specific; antiphagocytic) Other virulence factor: a.Protein F mediates epithelial cell attachment b.Lipoteichoic acid bacterial adherence to the respiratory epithelium

ENZYMES AND TOXINS PRODUCED BY S. pyogenes


1. HEMOLYSINS A. STREPTOLYSIN O - Oxygen labile; highly antigenic - responsible for subsurface hemolysis on BAP - cause lysis of WBC, platelets, tissue cells - ASO test detects recent

ENZYMES AND TOXINS PRODUCED BY S. pyogenes


B. STREPTOLYSIN S - Oxygen stable; non antigenic - responsible for surface hemolysis on BAP - it causes lysis of WBCs 2. DEOXYRIBONUCLEASES

ENZYMES AND TOXINS PRODUCED BY S. pyogenes


3. STREPTOKINASE - a protein that binds to plasminogen and activates the production of plasmin - it allows the bacteria to move from clotted area - it activates a host blood-factor that dissolves fibrin clots

ENZYMES AND TOXINS PRODUCED BY S. pyogenes


4. HYALURONIDASE - to separate the tissue and spread the organism 5. PYROGENIC (ERYTHROGENIC) TOXINS - serotypes A, B, C - exotoxin B (cysteine protease) degrades proteins

INFECTIONS AND DISEASES

1. PHARYNGITIS OR TONSILITTIS STREP THROAT

2. SCARLETT FEVER (SCARLATINA)


Results from a throat infection with a strain of S. pyogenes that carries a lysogenic bacteriophage. Acquired through respiratory droplet Cardinal signs: diffused red rash on the upper chest and spreads to the trunk and extremities and strawberry colored-tongue Caused by the release of erythrogenic toxin/strep pyrogenic exotoxin

2. SCARLETT FEVER (SCARLATINA)

2. SCARLETT FEVER (SCARLATINA)


DICKS TEST (ERYTHROGENIC TOXIN) - susceptibility test for scarlet fever - (+) erythema redness SCHULTZ CHARLTON REACTION - diagnostic test for scarlet fever - used to diagnostic present scarlet fever - (+) blanching phenomenon

3. SKIN INFECTIONS
CELLULITIS - is a diffuse, spreading infection of subcutaneous skin tissue characterized by a defined area of redness (erythema) and accumulation of fluid (edema)

3. SKIN INFECTIONS
ERYSIPELAS - is an acute infection and inflammation of the dermal layer of the skin characterized by painful reddish patches that enlarge and thicken with sharply defined edges - may also lead to necrotizing fasciitis and myositis galloping

3. SKIN INFECTIONS
RHEUMATIC FEVER ACUTE GLOMERULONEPHRITIS OR BRIGHTS DISEASE - inflammatory disease of the renal glomeruli; results from the deposition of antigen-antibody complexes.

DIAGNOSTIC TESTS

BACITRACIN DISK TEST


Differentiates S. pyogenes from other -hemolytic group Result: (+) any zone of inhibition Group C and G are also susceptible

SULFAMETHOXAZOLETRIMETHOPRIM TEST
Positive: Any zone of inhibition Negative: No Zone of inhibition (Group A and Group B)
SXT

SXT

L-pyrrolidonyl-napththylamide (PYR) TEST


Specific than Bacitracin test (+) bright/cherry red color (napththylamide) upon adding 0.01% cinnamaldehyde reagent (-) no color change Other PYR test positive

GROUP B STREPTOCOCCI
Part of the normal flora of female genital tract and lower GIT Nosocomially transmitted by unwashed hands of mother or healthcare personnel. Infection of fetuses and infants infection during passage through the colonized birth canal, and premature rupture of mothers membranes

GROUP B STREPTOCOCCI
On culture, it is grayish white mucoid w/ small zone of beta hemolysis Species: S. agalactiae Virulence factor: capsule (sialic acid signifiant component of the capsule) Enzymes: hemolysin, CAMP factor, neuraminidase, deoxyribonuclease, hyaluronidase and protease

DISEASES AND INFECTIONS


Pneumonia Neonatal Meningitis Neonatal sepsis Postpartum infection Osteomyelitis UTI endocarditis

DIAGNOSTIC TESTS

CAMP TEST
To differentiate S. agalactiae from other -hemolytic streptococci It uses -lysin producing strain of S. aureus or disk impregnated w/ lysin Result (+) arrowhead hemolysis near S. aureus growth /

HIPPURATE HYDROLYSIS TEST


S. agalactiae possesses the enzyme hippuricase Reagent: sodium + ninhydrin Results (+) Purple color after adding Ninhydrin rgt (indicates hippurate hydrolysis)

GROUP C STREPTOCOCCI
Main source of streptokinase; animal pathogen Species: S. equi, S. equisimilis, S. dysagalactiae, S. zoopedimicus

GROUP D STREPTOCOCCI
Enterococci: E. faecalis, E. faecium, E. avium, E. gallinarium Non enterococci: S. bovis, S. equinus Enterococci are not highly pathogenic; cocci in pairs and long chains

DIAGNOSTIC TEST

GROWTH IN BILE ESCULIN MEDIUM


Reagent: Esculin + 1-4% bile salt Result: (+) Black color (BOTH enterococci and non enterococci)

6.5% NaCl (nutrient broth base) test


Result: (+) turbididty enterococci (also PYR +) (-) non enterococci

LAP Test
Detects for the ability of the bacteria to hydrolyze Leucine-naphthylamide by enzyme leucine Positive: Development of a red aminopeptidase to -naphthylamide.
color within 1 minute after adding cinnamaldehyde reagent (S. Pneumoniae and S. pyogenes, Enterococcus, Pediococcus )

Negative: No color change or development of slight yellow color (Aerococcus and Leuconostoc)

NOTES
Enterococci (+) bile esculin, growth in 6.5% NaCl. PYR test and LAP test; vancomycin resistant Leuconostoc, Pediococcus, Globicatella, S. urinalis, Lactococcus (+) growth in bile esculin and 6.5% NaCl All enterococci isolated from human hydrolyze PYR

NOTES
Isolation of S. bovis from a blood culture may be the first indication that the patient has an occult tumor in the GI tract (GI cancer) 85% of Group B streptococci is (+) w/ growth in 6.5% NaCl but (-) in bile esculin test E. faecalis requires cyanocobalamin (B12) as growth factor.

GROUP F and G
Minute streptococci w/ wide zone of beta hemolysis Species: S. anginosus

VIRIDANS STREPTOCOCCI
Alpha hemolytic streptococci that lack the lancefield group antigens and do not fall on the criteria for S. pneumoniae, can be non-hemolytic. Oropharyngeal commensals Opporunistic pathogens of low virulence Glucans and dextrans enhance attachment to host cell surfaces

VIRIDANS STREPTOCOCCI
Laboratory tests: bile insoluble, optochin resistant, no growth in 6.5% NaCl, (-) inulin fermentation and PYR (-) Species: S. mutans, S. salivarius, S. mitis Infections: subacute endocarditis, dental carries (S. mutans)

ABIOTROPHIA
Pyridoxal-dependent or Vit B6 dependent and symbiotic streptococci Formerly known as nutritionally variant streptococci (NVS) Thiol compounds cystein, Vit B6 and pyridoxal This group of organism were found as satellites around an organism that produces pyridoxal E. coli, Klebsiella spp.,Enterobacter spp., yeasts

ABIOTROPHIA
They produce satellatism w/ Staphylococcus Opportunistic pathogens of low virulence Variable morphology gram (+) to gram (-) to gram variable; pleomorphic forms It causes 5-6% endocarditis

STREPTOCOCCUS SPP.

LABORATORY DIAGNOSIS

1. Gram Stain 2. Culture BAP, PEA, CNA, CAP For isolation of Group A streptococci from throat swab, BAP w/ SXT is used to suppress the growth of normal flora. To detect genital carriage of Group B streptococci during pregnancy, Todd Hewitt broth w/ antibiotics (gentamicin, nalidixic acid or colistin and nalidixic acis) is used to suppress vaginal flora.

Visualization of beta hemolysis is enhanced by anaerobic condition (BAP should be inoculated by stabbing the inoculating loop into the agar several times) CAP is incubated w/ 5-10% CO2 Abiotrophia will not grow on BAP or CAP unless pyridoxal (B6) is supplied. Streptococci has tendency to produce increase amount of lactic acid and other organic acid from fermentation of sugar resulting to acidic medium and eventually their growth is inhibited.

3. Susceptibility Test bacitracin disk test SXT test 4. CAMP and Hippurate test 5. Bile Esculin and Salt Tolerance Test 6. Serological Test a.Latex agglutination b.ASO titer test c.ELISA d.Co-agglutination

Streptococcus pneumoniae
Diplococcus/pneumococcus Gram (+) cocci in pairs, oval or lancet shape; facultatively anaerobe Causative agent of lobar pneumonia Considered part of the normal flora (25-50%) of the URT of preschool children Most common bacterial pneumonia in elderly as well as in patients w/ underlying disease.

Streptococcus pneumoniae
Most common cause of bacterial meningitis in adults Common isolate both as a pathogen and as a member of the normal respiratory tract. Principal virulence factor: antiphagocytic capsular polysaccharide type 3 strains Opsonization of the capsule renders the organism non virulent

Streptococcus pneumoniae
The capsule is antigenic and can be identified w/ appropriate antiserum; it is composed of hyaluronic acid. Alpha hemolytic colonies, mucoid, flattened with depressed center. dome-shaped, glistening, wet, mucoid young colonies coin w/ a raise rim old colonies (due to autolytic

Streptococcus pneumoniae
Dimple-shaped/donut-shaped colonies Enzymes: hemolysin, immunoglobulin A protease, neuraminidase and hyaluronidase

INFECTIONS/DISEASES PRODUCED

1. Pneumonia - pneumonia due to S. pneumoniae is not usually a primary infection but rather a result of disturbance of the normal defense barriers. 2. Meningitis 3. Otitis media 4. Bacteremia 5. Endocarditis 6. Peritonitis

Differential Test 1. Optochin Susceptibility/P disk Test Presumptive test Ethylhydroxycuprein hydrochloride BAP (sensitivity media) 2. Bile Solubility Confirmatory test Differentiates pneumococcus from viridans streptococci Sodium desoxycholate When a heavy suspension of pneumococcus is added to bile salt, the cloudiness of the broth clears after 3 hrs incubation.

Result

> Or = 14mm

(+) soluble

Differential Test 3. Neufeld Quellung Reaction The antipnemococcal serum is mixed w/ sputum, CSF and other sources along w/ methylene blue Examined under OIO

Result

SWELLING CAPSULE

4. Mouse Virulence 5. Inulin Fermentation Test

Death of the animal (+) Fermentation

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