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 agar plate

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 variationa.Mucoidb.Matte or rough – large amount of CHONc.Smooth/glossy – little amount of CHON

CLASSIFICATION OF STREPTOCOCCI

A. ACADEMIC/BERGEY’S CLASSIFICATION• BASED ON TEMPERATURE

1. PYOGENIC GROUP

•Will not grow neither on 10°C or 45°C•Produce pus; mostly β-hemolytic•Species: Streptococcus pyogenes

2. VIRIDANS GROUP

• Will grow at 45°C• 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 10°C and 45°C•Normal flora of human intestine•Species: E. faecalis

4. LACTIC GROUP

• Will grow on 10°C• Non hemolytic organism• Often found in dairy products• Species: S. lactis

CLASSIFICATION OF STREPTOCOCCI

B. SMITH AND BROWN’S CLASSIFICATION•BASED ON HEMOLYTIC PATTERN

1. ALPHA-HEMOLYTIC STREPTOCOCCI

• Partial/incomplete hemolysis of RBC around colony

• Green discoloration around colony

• Species: • S. pneumoniae

(green streptococci)

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)

CLASSIFICATION OF STREPTOCOCCI

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.

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: M-protein (type specific; anti-phagocytic)• 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. HEMOLYSINSA. STREPTOLYSIN O - “Oxygen labile”; highly antigenic - responsible for subsurface hemolysis on BAP - cause lysis of WBC, platelets, tissue cells - ASO test – detects recent infection w/ S. pyogenes

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)

• DICK’S 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” – rash fade

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 gangrene”

3. SKIN INFECTIONS

• RHEUMATIC FEVER

• ACUTE GLOMERULONEPHRITIS OR BRIGHT’S 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 β-hemolytic group

• Result: (+) any zone of inhibition

• Group C and G are also susceptible

SULFAMETHOXAZOLE-TRIMETHOPRIM TEST

• Positive: Any zone of inhibition

• Negative: No Zone of inhibition

• (Group A and Group B)

SXT

SXT

L-pyrrolidonyl-β-napththylamide (PYR) TEST

• Specific than Bacitracin test

(+) bright/cherry red color (β-napththylamide) upon adding 0.01% cinnamaldehyde reagent

(-) no color changeOther PYR test positive

organisms: Enterococcus, Aerococcus, Gemella

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 mother’s 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 β-hemolytic streptococci

• It uses β-lysin – producing strain of S. aureus or disk impregnated w/ β lysin

• Result (+) arrow-head β-hemolysis near S. aureus growth / (+) bowtie appearance

HIPPURATE HYDROLYSIS TEST

• S. agalactiae possesses the enzyme hippuricase

• Reagent: sodium + ninhydrin

• Results (+) Purple color after

adding Ninhydrin rgt (indicates hippurate hydrolysis)

(-) no color change

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-β-naphthylamide by enzyme leucine aminopeptidase to β-naphthylamide. Positive: Development of a red 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

LABORATORY DIAGNOSISSTREPTOCOCCUS SPP.

1. Gram Stain2. 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 test4. CAMP and Hippurate test5. Bile Esculin and Salt Tolerance Test6. Serological Test

a.Latex agglutinationb.ASO titer testc.ELISAd.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 changes)

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. Meningitis3. Otitis media4. Bacteremia5. Endocarditis6. Peritonitis

Differential Test Result

1. Optochin Susceptibility/P disk Test

• Presumptive test• Ethylhydroxycuprein

hydrochloride• BAP (sensitivity media)

> Or = 14mm

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.

(+) soluble

Differential Test Result

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

SWELLING CAPSULE

4. Mouse Virulence Death of the animal

5. Inulin Fermentation Test (+) Fermentation