Streptococcaceae and Enterococcus species Flashcards
Specimens to be obtained for culture depend on the nature of the streptococcal infection.
- Throat swab
- Pus
- Cerebrospinal fluid
- Blood
- Other sterile body fluid
Spherical or oval-shaped, however, rodlike forms are occasionally seen.
Streptococcus and Enterococcus: G+ cocci in chains
Tend to appear as chains of diplococcal cells rather than as chains of individual cells.
Streptococcus and Enterococcus: G+ cocci in chains
Broth cultures will show more chaining than those made from agar plates.
Streptococcus and Enterococcus: G+ cocci in chains
Cells have slightly elongated “lanceolate” or lancet-shaped morphology
Streptococcus pneumoniae : G+ cocci in pairs
“Halo” may be observed surrounding the organism pairs, indicating the presence of the polysaccharide capsule
Streptococcus pneumoniae : G+ cocci in pairs
Single cocci or chains are also seen in direct smears.
Streptococcus pneumoniae : G+ cocci in pairs
Culture media OF STREPTOCOCCI
- 5% Sheep BAM
- Columbia CNA
- PEA
Technique for inoculating throat swab for S. pyogenes: stabbing the inoculating loop into the agar after streaking the blood agar plate forces some inoculum under the agar, thereby creating a relatively anaerobic environment.
Streak-stab technique
Allows subsurface colonies to display hemolysis caused by O2-labile streptolysin O.
Streak-stab technique
Other methods: pour-plate, agar-overlay
Streak-stab technique
Growth is enhanced 5% to 10% CO2.
Streak-stab technique
Most will grow on agar media at
35-37 oC within 48 hours
About 0.5 mm in diameter, grayish white, convex, entire, transparent to translucent, matte or glossy; large zone of β-hemolysis, 2x-4x the diameter of the colony.
Group A streptococci
Larger than group A streptococci; translucent to opaque; flat, glossy; narrow zone of β-hemolysis
Group B streptococci
Grayish white, glistening; wide zone of β-hemolysis
Group C streptococci
Grayish white, small, matte; narrow to wide zone of β-hemolysis
Group F streptococci
Grayish white, matte; wide zone of β-hemolysis
Group G streptococci
Small, gray, glistening; young (18-24 h) are dome-shaped, later (after 24-48 h) developing central depression with elevated margin (umbilicated) giving the characteristic “checker piece”, “nail-head” or “draughtsman” appearance; if organism has a polysaccharide capsule, colony may be mucoid; α-hemolytic
S. pneumoniae
Minute to small, gray, domed, smooth or matte; α- or ɣ-hemolysis
Viridans streptococci
Small, cream or white, smooth, entire; α–, β-, or ɣ-hemolysis
Enterococcus spp.
More intense β-hemolysis is noted in areas where the medium has been “stabbed,” pushing some of the bacteria under the medium surface.
β-Hemolytic streptococci on sheep blood agar.
The hemolysis in these areas is due to the combined activities of streptolysin O and streptolysin S, the principal hemolysins of group A streptococci.
β-Hemolytic streptococci on sheep blood agar.
Streptolysin O is oxygen-labile and does not show maximal activity on the surface of the agar; the surface β-hemolysis is largely due to streptolysin S, which is oxygen-stable.
β-Hemolytic streptococci on sheep blood agar.
Partial hemolysis of the erythrocytes results in a “greening” of the agar medium surrounding the colonies (αhemolysis).
⍶-Hemolytic streptococci on sheep blood agar.
Streptococci that are α-hemolytic include S. pneumoniae, the viridans group of streptococci, and occasionally Enterococcus species.
⍶-Hemolytic streptococci on sheep blood agar.
Colonies ofEnterococcus speciescultivated on sheep blood, 24 hours in an aerobic atmosphere, 37°C, typically exhibits γ-hemolysison sheep’s blood agar, but some strains areα-hemolyticor evenβ-hemolytic.
γ-Hemolytic streptococci on sheep blood agar.
Other ɣhemolytic streptococci include group D streptococci (S. bovis), and sometimes viridans streptococci.
γ-Hemolytic streptococci on sheep blood agar.
Two characteristics of S. pneumoniae can be used for presumptive identification.
Streptococcus pneumoniae colonies on sheep blood agar.
On the left is shown a typical α-hemolytic, dome-shaped, mucoid strain of S. pneumoniae growing on sheep blood agar
Streptococcus pneumoniae colonies on sheep blood agar.
Its appearance is due to the production of large amounts of capsular polysaccharide.
Streptococcus pneumoniae colonies on sheep blood agar.
On the right is a close-up photograph illustrating the collapse of the central portion of the colonies owing to organism autolysis, resulting in the so-called checker-piece and nail-head colony morphologies shown here.
Streptococcus pneumoniae colonies on sheep blood agar.
This test classifies the gram-positive cocci into catalase-positive staphylococci and micrococci, and catalase-negative streptococci, enterococci, and the Streptococcus-like bacteria.
Catalase test
These tests are done on catalase-negative, gram-positive cocci, placing them in the streptococcus group, then initially may be classified on the basis of their hemolytic properties on sheep blood agar.
Identification tests
Used for presumptive identification of group A β-hemolytic streptococci (bacitracin-susceptible)
Bacitracin (Taxo A) test
Principle: Based on the selective inhibition of the growth of group A β-hemolytic streptococci by low concentration of bacitracin (0.04 U).
Bacitracin (Taxo A) test
Used for presumptive identification of groups C, F, or G βhemolytic streptococci; it must be performed in conjunction
with bacitracin test because groups C, F, and G streptococci
are also inhibited by bacitracin.
SXT (Trimethoprim-sulfamethoxazole) test
Based on the selective inhibition of the growth of [?] by trimethoprim (1.25 ug) and sulfamethoxazole (23.75 ug).
non-A and non-B β-hemolytic streptococci
SXT (Trimethoprim-sulfamethoxazole) test
Positive result [susceptible (S)]:
Any zone of inhibition around the
disk
SXT (Trimethoprim-sulfamethoxazole) test
Negative result [resistant (R)]:
No zone of inhibition
Bacitracin (S), SXT (R)
Group A streptococcus
Bacitracin (R), SXT (R)
Group B streptococcus
Bacitracin (S or R), SXT (S)
Group C, F, G streptococcus
The test was first described in 1944 by [?], and it is their names that provide the acronym (CAMP) for the test.
Christie, Atkins, and
Munch–Petersen
CAMP test is used for presumptive identification of
bacitracin-resistant Group B β-hemolytic streptococci
CAMP test is used for presumptive identification of
bacitracin-resistant Group B β-hemolytic streptococci
Based on the production of extracellular substance, CAMP factor, by group B streptococci that enhances the hemolytic activity of the β-hemolysin-producing strain of Staphylococcus aureus causing [?] observed as a arrow-head shaped zone of clearing in the area on BAM where the two organisms intersect.
“synergistic hemolysis”
CAMP test: Positive result
Arrowhead-shaped zone of β-hemolysis at the juncture of the two organisms.
CAMP test: Negative result
No arrowhead-shaped zone of β-hemolysis.
: CAMP-positive
: CAMP-negative:
Group B streptococcus
Group A streptococcus
Sodium hippurate hydrolysis test is used for presumptive identification of
bacitracin-resistant Group B β-hemolytic streptococci
Sodium hippurate hydrolysis test is based on the hippuricase activity of Group B streptococci that hydrolyzes hippurate to its components, [?].
benzoate and glycine
Test for benzoic acid:
Benzoic acid is precipitated by [?] that persists after 10 minutes.
If the precipitate clears within 10 minutes, it is due to nonspecific reaction of hippurate and other proteins which are also precipitated by FeCl3 but are [?] than benzoate in excess FeCl3.
ferric chloride (FeCl3)
more readily soluble
Test for benzoic acid: Negative result
Heavy precipitation that persists for 10 minutes or longer.
Test for benzoic acid: Positive result
Precipitation that clears within 10 minutes.
[?], a strong oxidizing agent, deaminates alpha-amino acid compounds (glycine) with the release of ammonia and carbon dioxide.
Ninhydrin
The released [?] reacts with the residual ninhydrin to form a [?] color.
ammonia
purple or deep blue
Test for glycine acid: Positive result
deep blue or purple color
Test for glycine acid: Positive result
colorless or slightly yellow- pink color
: Hippurate-positive
: Hippurate-negative
Group A streptococcus
Group B streptococcus
Optochin (ethylhydrocupreine hydrochloride), a quinine derivative, selectively inhibits the growth of S. pneumoniae at
very low concentrations (5 ug/mL or less).
Positive result (optochin-susceptible):
Negative result (optochin-resistant):
A zone of inhibition of >14 mm with 6-mm disc (>16 mm with 10-mm disc)
No zone of inhibition or a zone of inhibition <14 mm (or <16 mm)
Optochin (S)
Optochin (R)
Streptococcus pneumoniae
Viridans streptococci and group D enterococci
S. pneumoniae produces an autolytic, intracellular amidase enzyme that is activated by bile, [?], causing the organisms to undergo rapid autolysis.
desoxycholate
Positive result (bile-soluble)
Disappearance of colonies on which the reagent was placed; an imprint of the lysed colonies may remain within the zone.
Negative result (bile-insoluble)
Colonies where the reagent was placed remains intact and visible.
Bile-soluble
Bile-insoluble
S. pneumoniae
Viridans streptococci and group D enterococci
Based on the reaction of specific anticapsular antibodies with polysaccharide capsule (in S. pneumoniae, or other encapsulated bacteria) causing a [?] on the surface of the organism and a change in the refractive index of the capsule itself. Microscopically, the capsule appears to swell.
microprecipitation reaction
Quellung (+)
Quellung (-)
S. pneumoniae
Viridans streptococci
A small amount of growth is mixed with [?], antisera against the capsule polysaccharide, and [?].
After incubation at room temperature for [?], the reaction is observed under the microscope. The organisms are outlined in light blue.
A positive reaction shows [?] because of cross-linking of the antibodies and pneumococci. The halo effect around the pneumococci is apparent [?].
saline
methylene blue stain
1 hour
clumping
capsular swelling
Based on the ability of S. pneumoniae to [?].
ferment inulin with acid and gas production
Inulin (+)
Inulin (-)
S. pneumoniae
Viridans streptococci
Mouse virulence test is based on the sensitivity of white mouse to [?], that intraperitoneal injection of even a few of organisms (in sputum or CSF) will result in death of the mouse, usually within 1-2 days.
S. pneumoniae
death of the mouse w/in 1-2 days
negative
Streptococcus pneumoniae
Viridans streptococci
The tests are used for presumptive identification of group D streptococci (nonenterococci ) and enterococci, and to distinguish them from other .
ɣ-hemolytic streptococci
Bile-esculin test is based on the ability of certain bacteria, notably group D streptococci and Enterococcus species to grow in the presence of [?] and hydrolyze esculin into glucose and esculetin.
Esculetin, in turn, reacts with [?] (supplied by inorganic medium component ferric citrate) to form black diffusible complex.
40% bile (or 4% bile salts)
ferric ions
Positive result:
Negative result:
Blackening of the medium.
No blackening of the medium.
Bile-esculin-positive
Bile-esculin-negative
Group D streptococci / Enterococcus spp
Viridans streptococcus, not group D
Based on the specific ability of Enterococcus species to grow in the presence of [?] incorporated in either a broth or agar medium.
6.5% NaCl
Positive result (salt-tolerant): Negative result (non-salt-tolerant):
Visible turbidity in broth with or without color change from purple to yellow.
No turbidity and no color change .
salt-tolerant
non-salt-tolerant
Enterococcus species (E. faecalis, E. faecium) Group D streptococci (S. bovis and S. equinus)
Pyrrolidonyl Arylamidase (PYR) Test based on the hydrolysis of [?] by a specific aminopeptidase enzyme, pyrrolidonyl arylamidase.
Release of free β-naphthylamide is then detected by addition of the diazo dye coupler, [?] to form a red Schiff base.
PYR (L-pyrrolidonyl-β-naphthylamide)
N, N-dimethylaminocinnamaldehyde
PYR-positive
PYR-negative
Enterococcus species
Group D streptococcii
penicillin-resistant
penicillin-susceptible
Enterococcus species
Group D streptococci (nonenterococcus)
