MEDT 14 | CLIN. BACTERIOLOGY (LEC) LESSON 7.

FAMILY STREPTOCOCCACEAE
Jonnel P. Andaya || 3rd Year (GRAM-POSITIVE COCCI)
Transcribed by: De los Reyes, JD | Ybanez, TM

 Non-motile bacteria
OUTLINE:  In thioglycolate broth it reveals that streptococcaceae is a
facultative anaerobes
I. GENERAL CHARACTERISTICS
II. SPECIES MOSTLY ENCOUNTERED IN HUMAN SPECIES MOSTLY ENCOUNTERED IN HUMAN
INFECTIONS INFECTIONS
A. STREPTOCOCCUS PYOGENES ● Streptococcus pyogenes
B. STREPTOCOCCUS AGALACTIAE ● Streptococcus agalactiae
C. GROUP C, F, AND G BETA-HEMOLYTIC ● Streptococcus pneumoniae
STREPTOCOCCI ● Viridans Group of Streptococcus
D. STREPTOCOCCUS PNEUMONIAE ● Enterococci spp.
E. VIRIDANS STREPTOCOCCI
Streptococcus pyogenes (GROUP A)
F. ENTEROCOCCUS SPP.
● It can be a colonizer. Colonizer can be in normal flora BUT
G. LEUCONOSTOC SPP., LACTOCOCCUS rarely encountered as normal flora.
SPP., PEDIOCOCCUS SPP., GEMELLA SPP. ● It can be found in skin, mucosa, upper respiratory tract, and
H. MISCELLANEOUS OTHER GRAM POSITIVE pharyngeal.
COCCI ● It is always clinically significant
III. LABORATORY DIAGNOSIS ● It has many virulence factors:
A. DIRECT DETECTION METHOD o Protein F and Protein M (Antiphagocytic)
o They have the streptolysin which is the hemolysin
B. LATEX AGGLUTINATION AND ELISA FOR
(The streptococcus pyogenes in agar plate have a
S. PYOGENES distinguishing hemolytic pattern of Beta hemolytic.
C. ANTIGEN DETECTION KIT FOR GROUP B It has a complete clearing around the colonies
STREPTOCOCCUS (S. AGALACTIAE): because of hemolysin. They can cause hemolysis
D. LATEX AGGLUTINATION FOR GROUP B of red blood cells.
STREPTOCOCCUS o They produced 2 types of hemolysin:
IV. SPECIMEN PROCESSING  Streptolysin S- it is the oxygen stable
which is less antigenic or non-antigenic
V. SPECIMEN COLLECTION
 Streptolysin O – highly antigenic and
VI. MEDIA OF CHOICE AND CULTIVATION oxygen labile
VII. GRAM STAINING
VIII. APPROACH IN IDENTIFICATIONS Neutralization Inhibition Test - Streptolysins are
IX. CLASSIFICATIONS used to detect if the patient has a streptococcus
infection.
A. BROWN-SMITH CLASSIFICATIONS (BASED
ON PATTERN OF HEMOLYSIS)
B. LANCEFIELD CLASSIFICATION
● It has spreading factors:
(ACCORDING TO CARBOHYDRATES o DNase
COMPONENT) o Hyaluronidase
C. ACADEMIC/BERGEYS CLASSIFICATION o Streptokinase
(ACCORDING TO TEMPERATURE) o Pyrogenic toxin / erythrogenic toxin – it causes
X. SUMMARY LABORATORY IDENTIFICATION the patient with streptococcus infection have
erythema – disseminated rashes all over the body
A. IDENTIFICATION OF S. PNEUMONIA TO
● Mode of Transmission:
VIRIDANS GROUP o Patient to patient contact (contact with
B. IDENTIFICATION OF ENTEROCOCCUS TO contaminated body fluids and droplets from the
NON-ENTEROCOCCUS patient
C. IDENTIFICATION OF GROUP A, B, C, AND D ● Disease Associated:
STREPTOCOCCUS o Myositis
XI. PRINCIPLES OF LABORATORY TESTS o Rheumatic Heart Disease
A. BILE ESCULIN TEST
M protein is structurally similar with muscle fibers protein
B. SODIUM HIPPURATE TEST and heart muscle proteins. When the streptococcus
C. PYR TEST ( PYROLIDONYL pyogenes induced in the body and the antibodies
ACRYLAMIDASE TEST) stimulate, the antibodies may attack mistakenly the
XII. ANTIMICROBIAL THERAPY muscle fibers or heart muscle proteins. This result to
“autoimmune disease”
GENERAL CHARACTERISTICS
o Pneumonia
 They are gram-positive bacteria
o Scarlet fever - due to pyrogenic and erythrogenic
 They are in cocci in chains “Lancet-shaped” toxin
 Catalyst negative
 They are pin point colonies The spread of pyrogenic or erythrogenic toxin in the
 Alpha, Beta, Gamma hemolytic body and in blood causes Streptococcal toxic shock
syndrome. Post streptococcal implications are renal
 Mucoid
failure and respiratory failure
 Increase amount of M protein – mattle rough
 Decrease amount of M protein – smooth glossy o Erysipelas – infection of upper layers of the skin.
 Beta hemolytic streptococcaceae have butter like odor
PAGE 2 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | FAMILY MICROCOCCACEAE (GRAM-POSITIVE COCCI)
Cellulitis – infection of deep layer of skin (subcutaneous
tissue)
Erysipelas – more superficial layer of the skin,
marginated. It may cause tonsilitis in children and
pharyngitis in adult.
o Poststreptococcal Glomerulonephritis – after
infection of streptococcus, the body produced
antibodies not only against the bacteria but also
against kidneys.
o Necrotizing fasciitis – flesh eating bacteria cause
by invasive group A streptococcus
Streptococcus agalactiae (GROUP B)
● It is a normal flora of female genital tract, upper respiratory,
and gastrointestinal tract
● When isolated, usually they are not clinically significant
because they are normal flora BUT they will be clinically
significant if this bacteria are isolated among pregnant
women.
● Virulence factor:
o Capsular – antiphagocytic
● Disease associated:
o Ruptured amnionic sac
● Mode of transmission:
o Perinatal transmission (mother to baby)
Since neonates’ immune system are not fully
developed, they can acquire the infection which may
cause “neonatal sepsis”, shock, baby meningitis, and
death.
Early onset – symptoms can be observed after 5 days
Late onset – after 1 week through 3 months of age
Clinical manifestation of the mother could be
endometritis and pelvic abscess
In immune-compromised individuals like HIV
patients, may acquire sepsis in bloodstream and joints
which may also cause septic arthritis, myositis, and
endocarditis
Group C, F, and G beta-hemolytic Streptococci
● Group C are Streptococcus dysgalactiae, Streptococcus
equinus, and Streptococcus zooepidemicus
● They are normal flora of female genital tract,
gastrointestinal tract, and skin nasopharynx.
● Virulence factor
o Unidentified BUT similar to Streptococcus
agalactiae
● Disease Associated:
o Group C
 Acute Pharyngitis BUT very rare
o Group G
 Malignancies
Streptococcus pneumoniae
● Colonizers in nasopharynx of humans
● Normal flora of nasopharynx but is an opportunistic
pathogen
● Can be acquired through person to person spread or contact
with contaminated respiratory secretions
● For pneumonia, this occurs by aspiration of the organisms
into the lungs.
● Virulence factors:
1. Polysaccharides capsule
o it inhibits phagocytosis
2. Pneumolysis
o it has various effect on host cells. Several factors
are likely involved in eliciting a strong cellular
response by the host.
o It can cause disruption, destruction of epithelial
lining of alveolar cells
● Disease Associated:
o Leading cause of pneumonia (lobar pneumonia
with rusty sputum)
o Meningitis with or without bacteremia
o Sinusitis and otitis media
Viridans streptococci
● Normal flora of human cavity, gastrointestinal tract and
female genital tract.
● Endogenous strains gaining access to normally sterile site
most notably result from dental manipulation with transient
bacteremia.
● Viridans streptococci: S. mutans, S. imitis, S. salivarius, S.
anginosus
● Alloiococcus spp.: catalase negative; only when tested on
media, devoid of whole blood (Chocolate agar). It
morphologically resembles the Viridans streptococci
● Viridans streptococci and Abiotrophia spp. are generally
considered to be opportunistic pathogen.
● Virulence factors:
1. Generally low virulence
2. Cellular complex of polysaccharides
● Disease Associated:
1. Slowly evolving (subacute) endocarditis
2. Dental carries (S. mutans)
Enterococcus spp. (GROUP D)
● Found in soil, food, water, and as normal flora of animals,
birds and human.
● The species most commonly associated with infection
(Enterococcus faecalis, Enterococcus faecium) are normal
flora of the human gastrointestinal tract and female
genitourinary tract.
● It can be transmitted by person to person contact, and
directly or by contaminated medical equipment, allow
nosocomial spread colonization with multi-drug resistant
strains.
● This genus is intrinsically more resistant to the antimicrobial
agents commonly used in hospitals and is especially
resistant to all currently available cephalosporins and
aminoglycosides.
● This genus is the first clinically relevant group of gram
positive cocci to acquire and disseminate resistance to
vancomycin, the single cell –wall agent available for use
against gram positive organisms to beta lactams (e.i.
Methicillin resistant Staphylococci)
● E. faecalis and E. faecium are the species that commonly
encountered. Between these two species, E. faecalis is the
commonly encountered, but the incidence of E. faecium
infection is on the rise in many hospitals, which is probably
related in some way to the acquisition of resistance to
vancomycin.
● Most common species are Enterococcus faecium, and
Enterococcus faecalis, yellow motile strains of the
Enterococci. Some usually nonpathogenic (vancomycin-
resistant) includes: E.casiflavus and E. gallinarum
● Virulence factors:
1. Little known about virulence
2. Adhesion and cytolysis
3. Other metabolic capabilities may allow these organisms
to proliferate as nosocomial pathogen
● Alloiococcus otitidis: causes otitis media in children
● Non-group D (non-enterococcus): S. bovis is associated
with gastrointestinal malignancy and may be an early
detection of gastrointestinal cancer. It is cephalosporin and
aminoglycoside resistant.
● Disease associated:
o Most infections are nosocomial and include urinary
tract infections, bacteremia, endocarditis, mixed
infections of abdomen and pelvis, wounds and
occasionally ocular infections
Abiotrophia spp. (Nutritionally variant Streptococci)
PAGE 2 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | FAMILY MICROCOCCACEAE (GRAM-POSITIVE COCCI)
● Normal flora of human oral cavity
● Uncertain, probably by endogenous strains gaining access
to normally sterile sites
● Nutritionally variant stage: Granulicatella spp., S. adjacens
and S. defecticus
● Disease Associated:
o Endocarditis – rarely encountered in infections of
other sterile sites
o Immunocompromised patients are prone in
infections
Leuconostoc spp., Lactococcus spp., Pediococcus spp.,
Gemella spp.
● Leuconostoc spp. – found in plants, vegetables, dairy
products ; vancomycin-resistant
● Lactococcus spp. – found in foods and vegetation
● Pediococcus spp. – found in foods and vegetation
● Gemella spp. – found in human normal flora in oral cavity
and upper respiratory tract
● Most are transient colonizers of gastrointestinal tract after
eating
● It may cause infection when it gain access to sterile sites,
usually in immunocompromised patients
● Rarely associated with human infections
● Diseases associated:
o Endocarditis – rarely encountered
o Usually in immunocompromised patients
Miscellaneous Other Gram Positive Cocci
● Other genera is low virulence and is only involved in
immunocompromised patients except Alloiococcus otitidis
which causes otitis media in children.
LABORATORY DIAGNOSIS
DIRECT DETECTION METHOD
1. Latex agglutination and ELISA for S. pyogenes: These
methods may cause false negative in low number of S.
pyogenes. Sensitivity has ranged from 60% to 95%
depending on the methodology and several other variables.
Therefore many microbiologists recommend collecting 2
throat swab
2. Antigen detection kit for Group B streptococcus (S.
agalactiae): for diagnosis of neonatal sepsis and meningitis
caused by group b streptococcus. It is developed for testing
urine, blood and CSF sample but urine may produce false
positive result and the best sample to be used is CSF.
3. Latex agglutination for Group B streptococcus: Kits to
detect the capsular Polysaccharides antigen of the
pneumococcus have also been developed for use with
urine, serum and CSF although they are no longer
commonly use in clinical microbiology.
SPECIMEN PROCESSING
● No special considerations are required for processing the
organisms
SPECIMEN COLLECTION
● No special considerations are required for specimen
collection and transport of the organisms.
● When cultural vaginal/rectal swabs from pregnant women
specifically for group B Streptococci, specimen should first
be inoculated to a selective broth such as LIM or carrot broth
or on to selective agar
o Example: Granada Agar
MEDIA OF CHOICE AND CULTIVATION
● Chocolate agar and Blood agar exception to Abiotrophia spp
and Granulicutella spp.
● Will grow on selective media such as CAN (Columbia agar
with colistin and nalidixic acid) and PEA (phenylethyl
alcohol)
● Abiotrophia spp. and Granulicutella spp. will not grow on
blood agar and chocolate agar unless pyridoxal (vitamin B6)
is supplied either by placement of a pyridoxal disk or by
cross streak with Staphylococcus or by inoculation of
vitamin B6- supplemented culture media.
● Blood cultures that appear positive and show chaining gram
positive cocci on gram stain but do not grow on subculture
should be re-subculture with pyridoxal disk to cover
possibility of Abiotrophia spp. or Granulicatella spp.
Bacteremia
● Todd-Hewitt broth with antimicrobials (with nalidixic acid,
gentamicin, and colistin) is used to suppress vaginal flora
and allowe the growth of S. agalactiae following subculture
to blood agar.
● Most laboratories incubate blood or chocolate agar plate 5%
to 10% carbon dioxide. This is the preferred atmosphere for
S. pneumonia and is acceptable for all genera discussed in
this chapter. However, visualization of betahemolysis is
enhanced by anaerobic conditions.
● Colonies can then grow throughout the depth of the agar,
producing subsurface oxygen-sensitive hemolysis (i.e
Streptolysis ). Most organism will grow on agar media within
48 hrs of inoculation.
● Beta- hemolytic streptococci may have distinct butter odor
GRAM STAINING
● All the genera described in this module are gram positive
cocci.
● Microscopically streptococci are round or be oval shaped
occasionally forming elongated cells that resembles
pleomorphic Corynebacterium spp. and Lactobacillus spp.
● They may appear gram negative if cultures are old or if the
patient has been treated with antibiotics.
● S. pneumoniae is usually lancet-shaped and occurs-singly
in pairs or in short chains.
● Growth in broth should be used for determination of cellular
morphology if there is question regarding staining
characteristics form solid media. - In this module, genera are
subdivided on the basis of gram stain based on whether they
have a “staph-like- gram stain” or “strep-like gram stain”.
Abitrophia spp. growing in broth form long chains of cocci -
Growth in broth should be used for determination of cellular
morphology if there is question regarding staining
characteristics form solid media.
● Aerococcus, Gemella, and Pediococcus : grows large,
spherical cocci arrange in tetrads or as individual cells. -
Leuconostoc: may elongate to form coccobacilli, although
cocci are the primary morphology.
APPROACH IN CLLASSIFICATIONS
● If the presence of hemolysis is uncertain, the colony should
be moved aside with a loop and the medium directly beneath
the original colony should be examined by holding the place
in front of a light source.
● Screening test for vancomycin susceptibility is often useful
for differentiating among many alpha hemolytic cocci. All
Streptococci spp., Aerococci spp., Gemella spp., Lactococci
spp. and most Enterococci spp. are susceptible to
vancomycin, whereas Pediococci spp., Leuconoctoc spp.
and many Lactobacilli spp. are typically resistant.
● The PYR and Hippurate or Camp test can be use to identify
Group A and Group B streptococci.
● S. pyogenes is the only species of beta-hemolytic
streptococci that will give a positive PYR reaction. - S.
agalactiae is able to hydrolyze hippurate and is positive to
CAMP test. The Camp test. The Camp test detects
production of diffusible, extracellular protein that enhances
the hemolysis of sheep erythrocytes by Staphylococcus
aureus.
● Positive Camp Test is the appearance of an arrow head
shape at the juncture of the S. agalactiae and S. aureus
streak. Occasionally non-beta hemolytic strains of S.
PAGE 2 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | FAMILY MICROCOCCACEAE (GRAM-POSITIVE COCCI)

agalactiae may be encountered, but identification of such Tests S. pneumoniae Viridans Group
isolates can be accomplished using the serologic Mouse virulence + -
agglutination approach. Indole + -
● S. pneumonia must be tested for either bile solubility test or fermentation
susceptibility to optochin (Taxo P) - Confirmatory to S. Bile solubility + -
pneumonia is bile solubility test; base on the ability of bile Optochin S R
salts to lyse Neufeld quelung + -
● Presumptive identification of S. pneumoniae is optochin test,
a filter paper disk impregnated with optochin is placed on a IDENTIFICATION OF ENTEROCOCCUS TO NON-
blood agar plate. ENTEROCOCCUS
● S. pneumoniae produces zone of inhibition, whereas
Viridans streptococci grow up to the disk - S. Group D PYR 6.5 % NaCl
pseudopneumoniae may interfere with appropriate Streptococcus
interpretation of the optochin disk Enterococcus + +
● S. pseudopneumoniae are resistant to optochin when they spp.
are incubated under increase CO2 (< 14mm) but are Non- - -
susceptible optochin (>14) when they incubated in ambient Enterococcus
atmosphere. spp.
● Once S. pneumoniae have been rule out as a possibility for
an alphahemolytic isolate, Viridans streptococci and IDENTIFICATION OF GROUP A, B, C, AND D
enterococci. Carbohydrates fermentation test are performed STREPTOCOCCUS
in heart infusion broth with bromcresol puple as an indicator.
Bacterial Group Taxo A SXT Other
CLASSIFICATIONS (Bacitacin) specific
test
BROWN-SMITH CLASSIFICATIONS (BASED ON PATTERN Group A Susceptible Resistant PYR (+)
OF HEMOLYSIS) Group B Resistant Resistant Camp test
(+),
1. Alpha - hemolytic: Partial lysis of RBC around colony; Hippurate
(+)
Greenish discoloration of area around colony Group C Resistant Susceptible
o Alpha hemolytic streptococci are: Streptococci Group D Resistant Resistant PYR and
pneumoniae and Viridans group of Streptococci (Enterococcus spp. ) 6.5% NaCl
(+)
2. Beta-Hemolytic: Complete hemolysis of RBC around Group Resistant Resistant PYR and
colony, clear are around colony (NonEnterococcus 6.5% NaCl
o Beta-hemolytic streptococci are: Group A, B, C and spp. ) (-)
some group D
3. Gamma-hemolytic: also known as non-hemolytic or no PRINCIPLES OF LABORATORY TESTS
hemolysis of rbc around colony, no change in agar 1. Bile esculin test:
o Gamma hemolytic: Mostly Enterococcus (Group D o Detects if organism can grow or survive in the
Streptococcus) presence of 90 % bile and can hydrolyze esculine.
(+) result: Brown black Precipitate
LANCEFIELD CLASSIFICATION (ACCORDING TO (+) organisms: Group D streptococcus
CARBOHYDRATES COMPONENT) 2. Sodium Hippurate Test:
o Determine if an organism ca hydrolyze hippurate to
1. Group A Streptococcus: S. pyogenes form 2 products : Glycine and benzoic acid.
2. Group B Streptococci: S. agalactiae (+) result: Purple color
3. Group C Streptococci: S. dysagalactiae, S. equimilis, and (+) organisms: Group B streptococcus
S. zooepidimicus, 3. PYR test ( Pyrolidonyl acrylamidase test):
4. Group D Streptococcus: Enterococcus spp. Non- o Rapid test for presumptive identification of group A
enterococci: S. bovis and S. equinus Streptococcus. Test to differentiate beta hemolytic
streptococcus; Substrate: L-pyrolidonyl B-
ACADEMIC/BERGEYS CLASSIFICATION (ACCORDING TO naphthylamine; Enzyme: L- pyrolydonyl
TEMPERATURE) aminopeptidase.
(+) Result: Red color
1. Pyrogenic Group: do not grow at 10 degrees Celsius and (+) Organisms: enterococcus
45 degrees Celsius and grows only at 37 degrees Celsius.
Mostly beta Hemolytic; DIAGNOSTIC TESTS
o example: S. pyogenes 1. Dicks test: Susceptibility tests
2. Viridans group: will grow at 45 and 37 degrees Celsius; not 2. Schultz-Charlton-Test: (+) result is blanching /pale
part of lancefield group; alpha hemolytic; phenomenon; neutralization tests
o examples: S. salivarius, S. mutans, S. imitis and S.
sanguis ANTIMICROBIAL THERAPY
3. Enterococcus group: will grow at 10 degrees, 45 degrees S. PYOGENES
and 37 degrees. Normal flora of human intestine; Penicillin is drug of choice; alternatives may include macrolides(
o example E. faecium and E. faecalis Azithromycin, Clarithromycin, Telithromycin) and
4. Lactic Group: will grow at 10 degrees and 37 degrees cephalosporins, vancomycin for penicillin allergic patients with
Celsius. It is non hemolytic organisms and often found in serious infection
dairy products;
o example: S. lactis ( normal coagulation or souring S. AGALACTIAE
of milk. Penicillin, ceftriaxone or cefotaxime, vancomycin- used for
penicillin allergic patients
SUMMARY OF LABORATORY IDENTIFICATION
IDENTIFICATION OF PNEUMONIA TO VIRIDANS GROUP GROUP C, F AND G BETAHEMOLYTIC STREPTOCOCCI
Penicillin and vancomycin
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S. PNEUMONIAE
Penicillin, Ceftriaxone, Cefotaxime, Telithromycin, trimethoprim
sulfamethoxazole, and certain quinolones (Levofloxacin,
ciprofloxacin, gemifloxacin)

VIRIDANS STREPTOCOCCI
Penicillin or ceftriaxone; vancomycin- is used in cases of
penicillin allergies and beta lactam resistance

ABIOTROPHIA SPP.
Penicillin, or vancomycin, plus aminoglycosides

ENTEROCOCCUS SPP.
For life threatening infection- penicillin, vancomycin plus
aminoglycosides; For multidrug resistance- Chloramphenicol;
For urinary tract infection-ampicillin, nitrofurantoin, tetracycline,
or quinolones