The Streptococci

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The Streptococci

• With Emphasis on Streptococcus and Enterococcus

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Preliminary Grouping of Gram Positive Cocci

• Gram Positive Coccus

Note SBA hemolysis as alt to PYR
Catalase

_
chains
_
Staphylococcus

Micrococcus
Rothia
PYR
Other Strep Group genera
_
See Staph PP

A Disk
S
R
Streptococcus sp other Group genera
S.pyogenes GAS A disc contains bacitracin
Enterococcus
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Strep Group GeneraNOT on the test

• Streptococcus
• Enterococcus
• Aerococcus
• Leuconostoc
• Abiotrophia
• Pediococcus
• Lactococcus
• Granulicatella
• Other new genera

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Metabolism

• Streptococci divide to form pairs and chains.
  Chain length varies among species depends upon
  growth conditions - broth generally favors chain
  formation
• The streptococci are catalase-negative
  Gram-positive cocci
• Streptococci conduct fermentation exclusively
  therefore grow fairly slowly. As strict
  fermentors their O2 requirement is described as
  aerotolerant anaerobes. Some grow better in the
  absence of oxygen but virtually all streps will
  grow anywhere in regard to O2
• Streptococcal colonies are usually 1 mm or less
  after 24h incubation under optimum conditions
  although this varies from species to species.

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Classification

• Genetic analysis indicated a lack of relatedness
  between some organisms previously in the Genus.
  The original genus Streptococcus has now been
  split into more than a dozen genera.
• Most notable of these were a few bile-resistant
  strep-like bacteria that inhabit the intestine of
  humans and many warm blooded animals
  Streptococcus faecalis and Streptococcus faecium.
  
• The Genus Enterococcus was created for these
  entericStreps hence E. faecalis and E. faecium
• Fortunately for clinical microbiologists and
  physicians the majority of the newly named
  genera contain few bacteria that are clinically
  significant
• The original genus Streptococcus and the genus
  Enterococcus comprise most of the human clinical
  isolates

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Classification

• Below are some newly named genera that were split
  from the genus Streptococcus
• Lactococcus
• Aerococcus
• Leuconostoc not important for test
• Pediococcus
• Gamella
• To reiterate these are rarely of clinical
  significance but are occasionally misidentified
  as Streptococcus or Enterococcus

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Classification - hemolysis

• In 1903 J.H. Brown grouped streps by their
  ability to lyse RBCs
• Beta hemolysis( ) complete lysis of RBCs
• Alpha hemolysis ( ) K removal from RBCs
  resulting in an opaque greenish-brown zone around
  colonies
• Gamma hemolysis ( ) no effect on RBCs
• Alpha prime hemolysis () small zone of intact
  RBCs adjacent to the colony surrounded by a zone
  of complete hemolysis
• Using the term gamma hemolysis is synonymous
  for having no effect - clinical microbiologists
  also call these organisms non-hemolytic
  streptococci

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Classification - Sherman

• Sherman in the early 1900s placed the streps in
  physiologic groups via physiological
  differences. His pyogenic group (to form pus)
  contained most of the pathogenic penicillin
  sensitive beta hemolytic species usually
  isolated from pus.
• Another physiologic group was the bile tolerant
  penicillin resistant gut bacteria removed from
  the genus Streptococcus and later becoming the
  genus Enterococcus
• It was he who reclassified Diplococcus pneumoniae
  (later officially changed to Streptococcus
  pneumoniae in the early 1970s) as the
  pneumococcus only 1 species. This organism
  was hemolytic originally penn sensitive
• It is (or at least was) by far the most common
  cause of community and hospital acquired
  bacterial pneumonia in humans. It is definitely
  the 1 cause of acute typical bacterial pneumonia.

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Classification - Sherman

• Another of Shermans groups included the alpha
  hemolytic bile and penicillin sensitive
  streptococci which are normal microbiota of the
  oral cavity several species are included here
  including S. mutans and S. sobrinus
• He named them the viridans group because of
  their tendency to produce alpha hemolysis.
  Viridian is latin for a green tint.

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Classification - Lancefield

• In 1927 Rebecca Lancefield discovered the ability
  to group streptococci based upon differences in
  streptococcal cell wall polysaccharides and the
  production of serum antibody in rabbits
  innoculated with these streptococcal antigens.
• Conducting precipitin tests in capillary tubes
  Lancefield combined known serum antibodies with
  extracted streptococcal antigen and identified
  which antigen(s) each species possessed.
• In this way Lancefield originated the
  Lancefield groups of Streptococcus that we
  still use to this day. For example
• S. pyogenes in Group A hemolytic
• S. agalactiae in Group B hemolytic
• S. zooepidemicus in Group C hemolytic
• S. faecalis in Group D variable

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Lancefield Capillary Precipitation
Rabbit Anti- serum
Ag-Ab interface
Ag-Ab interface
Rabbit Anti- serum
Strep Antigen Extract
No Precipitate (Negative Test)
Antibody against a strep group antigen
Precipitate (Positive Test)
Strep Antigen Extract
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Classification - Lancefield

• Lancefield realized that all species in each
  group generally (and conveniently) shared
  clinically significant properties such as type of
  hemolysis normal host body system or tissue
  where indigenous etc. For example
• Group A - S. pyogenes human upper respiratory
• Group B - S. agalactiate human urogenital
• Group C - S. zooepidemicus from animal products
• Group D - S. faecalis bile-resistant fecal
  origin
• Lancefield identified many other antigens and
  proposed several Lancefield groups. Groups A B
  C D F and G were the primary groups likely
  from human infections
• Lancefield later determined that viridans streps
  pneumococci did NOT possess antigens that
  reacted with her antisera
• More recently a new species S. milleri was
  found to carry AC F G antigens and display
  all 3 types of hemolysis.

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Preliminary Grouping NOT on test
Broth ch tet - - - - - -

Beta hemolysis /- - b -
- - - -
NaCl - /- /- /- -
45oC /- - - - -
Va S S b S S R R S
PYR -a -b - -
LAP - - /-
Streptococcus Enterococcus Lactococcus Aerococcus
Leuconostoc Pediococcus Gamella
Brothgrowth in broth chchains tettetrads
Vavancomycin PYR pyrrolidonyl arylamidase
LAPleucine aminopeptidase NaClgrowth in 6.5
sodium chloride Growth at 45oC a Streptococcus
pyogenes is PYR positive b Occasional exception
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Cultural Characteristics

• Streptococcus species tend to be more fastidious
  than staphylococci and Enterococcus
• SBA is ideal for growing Streptococcus as it
  contains various amino acids vitamins
  minerals. Any Additional catabolizable carbs
  present in SBA can mask the normal appearance of
  hemolysis making it appear
• Sheeps blood has 3 advantages over other animal
  blood
• 1. Haemophilus species will not grow on SBA due
  to the presence of NADase which neutralizes
  extracellular NAD (V factor).
• Hemolytic Haemophilus colonies on blood from
  other animals is easily confused with beta
  hemolytic Streptococcus species
• 2. Sheeps blood gives a distinctly clear beta
  hemolysis that is obviously different from alpha
  hemolysis not necessarily true of reactions on
  other animal bloods
• 3. Human blood can contain growth inhibiting
  antibodies etc that prevent recovery of
  organisms from samples

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Cultural Characteristics

• Growth of Streptococci can be enhanced by
  incubation in a CO2 rich environment (5-8).
  Some strains particularly some S. pneumoniae
  will not grow at all without added CO2
• Some streptococci grow better anaerobically than
  under any other conditions but remember that
  streps are aerotollerant
• As said earlier streptococci are fermentors and
  therfore form small colonies (some species form
  pin-point colonies)
• Most species form colonies that are translucent
  convex shiny
• S. pneumoniae colonies vary. Some have slightly
  raised edges to form a low convex colony others
  have a bump in the center (umbonate) and others
  have a smooth dip in the center (a concave or
  umbilicate colony)
• S. pneumoniae may have large capsules making
  their colonies mucoid these rarely form
  umbilicate colonies

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Cultural Characteristics

• Enterococcus species and Streptococcus agalactiae
  (group B streptococci or GBS) tend to grow larger
  colonies (and therefore more opaque) than other
  Streptococcus species
• Colonies of Enterococcus and GBS range form
  1mm-1.5mm in diameter in 18-24h

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Identification

• Streptococcal ID is important due to the
  potential severity of the condition but streps
  are fortunately fairly easy to ID to a point
• Presumptive ID can be performed with a few
  conventional tests. Isolation from any normally
  sterile body fluid or cavity necessitates
  definitive ID usually by technical means such as
  serotyping NA homology etc.
• The Streptococcus milleri group the new
  strep carries A C F and G antigens (47 of
  isolates type out as group F) or it may possess
  no Lancefield antigen at all. Also isolates can
  be either beta alpha or nonhemolytic
• The name S. milleri has been challenged by
  researchers at the CDC who had call it
  Streptococcus anginosus
• Whatever name is finally assigned to it this
  bacterium is definitely pathogenic and should be
  identified completely whenever isolated from
  normally sterile sites. SO how to do it

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Identification S. milleri

• Isolated from purulent deep tissue abscesses
• Pinpoint colony which is often beta hemolytic but
  could be non-hemolytic or alpha hemolytic
• Distinct caramel / butterscotch odor - variable
• Lancefield variable or non-groupable (47 F)
• Bacitracin resistant (vs S. pyogenes)
• Sulphonamide resistance (vs most other streps)
• Optochin resistance (vs alpha hemolytic streps)
• Tom VP positive

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Identification - NVS

• Another distinctive group of streptococci are
  Nutritionally Variant Strep (NVS) sometimes
  isolated from blood
• This group cannot synthesize certain growth
  factors (e.g. vitamin B6 and cysteine) which must
  be added to the culture medium. Common SBA does
  not contain these growth factors
• Detection of NVS can be accomplished by streaking
  SBA and then making a band streak of a Staph
  aureus culture across the plate (this is known as
  the Staph streak)
• NVS will grow as tiny colonies adjacent to the
  staph streak S. aureus synthesizes and secretes
  the required growth factors
• Commercial supplemented blood agar media (such as
  Danish blood agar) contains the required growth
  factors for NVS
• Gram positive catalase negative cocci that do
  not grow on sheeps blood or chocolate agar
  should be suspected and tested using the Staph
  streak test

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Staph Streak For NVS
Specimen streaked For isolated colonies
Staph aureus culture Band streak
NVS colonies Satelliting the Staph streak
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Identification - catalase

• The catalase test generally differentiates the
  two major groups of Gram-positive cocci (staph
  and strep) but it might not be straight forward
  as some streps produce pseudocatalatase
  especially when grown on SBA. This is
  demonstrated as slight bubbling in the presence
  of hydrogen peroxide after a brief delay.
• Care must also be taken when growth is collected
  from SBA because blood itself contains catalase.
  All in all it is better to grow cultures on
  non-blood media for catalase testing
• Cultures grown on SBA that show the
  characteristic pseuodocatalase reaction should
  be sub-cultured on non-blood media usually an
  agar slant. Drop H2O2 directly on the growth on
  the slant and monitor for bubbling. This is
  referred to as the tube catalase test.
• In a truly positive catalase test such as staph
  there is an immediate appearance of very obvious
  bubbling

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Presumptive ID Tests - CAMP

• The CAMP test (acronym for developers) is used
  to differentiate S. agalactiae (GBS) which is
  positive from other beta hemolytic strep all of
  which are negative
• CAMP factor is a soluble hemolysin produced by
  GBS that combines in a synergistic way with a
  similar hemolysin of S. aureus to form an
  arrowhead zone of clearing. See image
• The staph is streaked perpendicularly to the
  unknown strep. After appropriate incubation time
  an arrowhead-shaped clear zone of hemolysis
  will appear

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Presumptive Identifying Tests

• Several of the tests used to presumptively ID
  strep are sensitivity tests for certain
  antibiotics (bacitracin novobiocin
  sulfomethoxizole optochin etc.).
• The bile-esculin test (several formats) which
  determines if organisms can hydrolyze esculin (to
  glucose) in the presence of bile and the NaCl
  tolerance test both indicate Enterococci which
  are .
• The PYR (pyrrolidonyl peptidase) test is a rapid
  disk test performed on colonies growing on a agar
  media. Beta hemolytic GAS and Enterococcus are

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Presumptive ID NOT on test
Hemo- lysis a b
Baci- tracin S R b R b NA NA
NA NA
Bile Esculin - - -
- b -
Opto- chin NA NA NA R R
R S
Organism GAS GBS Groups CFG Entero- coccus Gro
up D Strep Viridans Pneumo- cocci
SXT R R S NA NA NA NA
CAMP - - - - - -
PYR - - - - -
NaCl - - b - - - -
NANot applicable a Weak beta b occasional
exception
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Antigen Testing

• Lancefield grouping can be used to ID strep as
  already discussed
• The original capillary precipitation test is
  tedious and time consuming but recently
  developed methods utilize inert particles coated
  with group specific antibodies
• Antigens extracted from unknown streptococci are
  mixed with the antibody coated particles and
  observed for agglutination
• Unlike Lancefields difficult extraction
  technique of boiling 24h broth cultures in an
  acid solvent recently developed extraction
  methods use enzymes or acid and take only
  minutes.
• GAS (from throat swabs) and GBS (colonies grown
  from vaginal or rectal swabs) antigens are
  sometimes detectable directly in clinical
  specimens or from enrichment broth cultures
• This GAS test is called the rapid strep test.

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Clinical Significance

• S. pyogenes (GAS) causes several diseases some of
  which have been previously discussed
• Streptococcal pharyngitis ( otitis media
  sinusitis)
• Scarlet fever
• Rheumatic fever
• Necrotizing fasciitis
• Streptococcal toxic shock syndrome
• Meningitis
• Glomerulonephritis
• Impetigo
• Cellulitis
• and more

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Clinical Significance

• S. agalactiae (GBS)
• Neonatal meningitis / septicemia
• Cystitis and pyelonephritis
• Postpartum endometritis
• Postpartum septicemia
• Suture site infections in women following
  caesarian section

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Clinical Significance

• Streptococcus pneumoniae
• Community acquired typical bacterial pneumonia
• Meningitis (any age group but predominates in
  adults over 60)
• Sinusitis and otitis media
• Bacteremia and septicemia (usually associated
  with meningitis and pneumonia)

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Clinical Significance

• Enterococcus sp.
• Upper and lower urinary tract infections (mainly
  nosocomial nursing homes)
• Native valve and prosthetic valve endocarditis
• Intra-abdominal and pelvic infections
• Wound infections
• Septicemia and meningitis in neonates and rarely
  other age groups

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Clinical Significance

• Beta hemolytic groups C F and G
• Recent reports suggest these organims may be
  emerging pathogens connected to various
  pathologies
• Acute pharyngitis
• Acute otitis media
• Endocarditis
• Meningitis
• Toxic shock syndrome
• Rheumatic fever
• Catheter-related septicemia

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Clinical Significance

• Viridans streptococci
• A leading cause (1 strep) of bacterial native
  valve and prosthetic valve endocarditis not
  pre-empted by streptococcal pharyngitis
• Dental caries capsule allows the bacterium
  (mainly S. mutans S. sobrinus) to adhere firmly
  to the teeth. Adherent bacteria produce acid
  fermentation products that dissolve the enamel of
  the teeth

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Clinical Significance - other

• Streptococcus milleri (S. anginosus) is
  associated with deep tissue and intra-abdominal
  abscesses
• The nutritionally variant streptococci (NVS) are
  associated with bacterial endocarditis

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Antimicrobial Susceptibility

• The antibiotic of choice for treating most
  streptococcal infections has always been
  penicillin G.
• GAS and GBS are uniformly susceptible to it
  although GAS penicillin resistance is present and
  growing
• Until recently virtually all S. pneumoniae were
  also susceptible to penicillin G. A small
  percentage has developed complete pen resistance
  and others are intermediately resistant
  (requiring a high dose of penicillin G to effect
  a cure)
• Enterococcus sp. are often moderately or
  completely resistant to penicillin G and other
  beta lactam antibiotics (Keflex etc)
• Until recently the drug of choice was ampicillin
  in enterococcal UTIs non-urinary infections -
  this resistance is increasing

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Antimicrobial Susceptibility

• Vancomycin is now the drug of choice for
  beta-lactam resistant Enterococcus species
• Very recently however even vancomycin resistance
  Enterococcus (VRE) have been detected in hospital
  outbreaks including Centra Health facilities
• Surveillance for VRE is conducted in all
  accredited medical facilities.
• A higher percentage of E. faecium isolates are
  VR than are E. faecalis isolates (E. faecium is
  the 1 most VR bacterium) however E. faecalis is
  a much more common human clinical isolate so
  there is no agreement as to which is worse.
• Presently there does not seem to be a good
  antimicrobial alternative available for treating
  serious infections caused by VRE

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Virulence Factors - GAS

• Capsule anti-phagocytosis specific attachment
  to specific tissues
• M protein in cell wall anti-phagocytosis
  specific attachment to specific tissues
• Streptolysin O and S leukocidins vs neutrophils
  macrophages
• Streptococcal pyrogenic exotoxins (SPE)
  superantigens directly stimulate T-cells
  (1000X more than a normal immune response) and
  cause them release high levels of the cytokines
  that are responsible for fever and shock
• SPE is also responsible for various tissue
  destruction including skin lesions and perhaps
  scarlet fever
• Hyaluronidase dissolves the hyaluronic acid that
  holds cellular basement membranes together
  (Hyaluronidase is therefore a spreading factor)

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Virulence Factors

• S. pneumoniae
• Capsule anti-phagocytosis specific attachment
  to specific tissues
• Pneumolysin anti-phagocytosis it also
  interferes with the action of tracheal cilia
  results in a croup-like cough
• Other GBS
• Capsule anti-phagocytosis specific attachment
  to specific tissues
• Enterococcus (some strains)
• Intrinsic resistance to beta lactam antibiotics
  due to the production of beta lactamases