Streptococcus and Enterococcus

1
Streptococcus and Enterococcus

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

2
Taxonomy of Streptococcus and Enterococcus

• Enterococcal group D Streptococcus (previously
  Streptococcus faecalis and Streptococcus faecium)
  currently classified in the genus Enterococcus
• Group D Streptococcus consists of a single
  pathogen Streptococcus bovis

3
Taxonomy of Streptococcus and Enterococcus

• Nutritionally-variant (pyridoxal-dependent)
  Streptococcus (NVS) currently classified in the
  genera Abiotrophia (A. defectiva) and
  Granulicatella (G. adiacens and G. elegans)

4
Hemolytic Reactions on Sheep Blood Agar

• Tryptic (trypticase) soy agar base with 5 sheep
  blood widely used for the determination of
  hemolytic reactions of Streptococcus and
  Enterococcus.
• The base medium contains soybean and casein
  peptides as a carbon and nitrogen source, and
  sheep blood provides hemin (X factor) for growth
  of fastidious organisms.

5
Hemolytic Reactions on Sheep Blood Agar

• Absence of carbohydrate prevents fermentative
  acids that inhibit hemolysis.
• Sheep blood contains NADase that inactivates NAD
  released by red cells, thus preventing growth of
  the ?-hemolytic organisms Haemophilus
  haemolyticus and H. parahaemolyticus that require
  exogenous NAD.

6
Hemolytic Reactions on Blood Agar (Sheep, Horse,
Rabbit)

• ?-hemolysis
• Complete clearing of blood agar due to lysis of
  red cells by oxygen-stable and oxygen-labile
  hemolysin
• ?-hemolysis
• Greening of blood agar due to partial lysis of
  red cells
• ?-hemolysis
• Absence of hemolysis

7
Streptococcal ?-Hemolysins

• Oxygen-labile streptolysin
• (Streptolysin O)1
• Oxygen-stable streptolysin
• (Streptolysin S)
• 1Activity of streptolysin O expressed in deep
  stabs of inoculum into anaerobic portions of
  sheep blood agar

8
(No Transcript)
9
(No Transcript)
10
Broad Groups of Streptococcus

• ?-hemolytic groups A, C, and G streptococci form
  large (gt0.5 mm) or small (lt0.5 mm) colonies on
  sheep blood agar.
• Large colony formers are pyogenic streptococci
  including Streptococcus pyogenes (group A)

11
Broad Groups of Streptococcus

• Small colony formers of ?-hemolytic streptococci
  with group A, C, or G antigens differ genetically
  from the pyogenic streptococci and are placed in
  the anginosus group of Streptococcus
• All ?-hemolytic group B streptococci are
  Streptococcus agalactiae

12
Broad Groups of Streptococcus

• Non-? hemolytic streptococci show either green
  hemolysis (?-hemolysis) or no hemolysis on sheep
  blood agar.
• A useful definition of viridans streptococci is
  organisms that demonstrate ?-hemolysis or no
  hemolysis.

13
Broad Groups of Streptococcus

• Viridans streptococci are grouped into
  Streptococcus pneumoniae, S. bovis, the anginosus
  group of Streptococcus, and other.
• Other groups of viridans streptococci (mitis,
  mutans, and salivarius) consist of
  endocarditis-producing strains, invasive strains
  in neutropenia, saprophytes, and contaminants.

14
Major Enterococcus Species

• Enterococcus faecalis constitutes 90 of
  clinically significant enterococcal isolates, E.
  faecium 10. Other species of Enterococcus
  rarely seen clinically.
• E. faecium demonstrates ?-hemolysis on sheep
  blood agar, E. faecalis no hemolysis with
  occasional strains (3) ?-hemolysis.

15
Pathogenic Species

• Streptococcus pyogenes (group A streptococci)
• Streptococcus agalactiae (group B streptococci)
• Streptococcus dysgalactiae subspecies equisimilis
  (group C and G streptococci)
• Streptococcus pneumoniae
• Streptococcus bovis

16
Pathogenic Species1

• Anginosus group of viridans streptococci (S.
  anginosus, S. constellatus, S. intermedius)
• Abiotrophia and Granulicatella species
• Enterococcus faecalis
• Enterococcus faecium
• 1Variably pathogenic

17
Saprophytic Species Infrequently Causing Disease

• Viridans streptococci other than S. pneumoniae,
  S. bovis, and species of the anginosus group
• Aerococcus, Gemella, and Stomatococcus1
• Species of Leuconostoc and Pediococcus2
• 1Enterococcus and Staphylococcus look alikes
• 2Uniform high-level vancomycin resistance

18
Streptococcus Natural Habitats

• Streptococci are resident colonizers of mucous
  membranes of the mouth, nasopharyngeal cavity,
  gastrointest-inal tract, and urogenital tract
• Streptococci are transient colonizers of the skin

19
Enterococcus Natural Habitats

• Enterococci can survive in harsh environments
  (extremes of temperature, salinity, and pH) and
  are ubiquitous in soil, water, plants, animals,
  birds, and insects
• Enterococcus faecalis and E. faecium normally
  inhabit the gastrointestinal and female genital
  tract

20
Streptococcus Modes of Infection

• Person to person transmission by direct contact
  with or coughing/sneezing of infected secretions
  followed by pharyngeal colonization and/or
  infection (Streptococcus pyogenes and S.
  pneumoniae)
• Colonization of the maternal genital tract by
  Streptococcus agalactiae with neonatal infection

21
Streptococcus and Enterococcus Modes of Infection

• Bacteremic dissemination from infected
  gastrointestinal malignancies (Streptococcus
  bovis)
• Endogenous strains gain access to sterile sites
  (groups C, F, and G ?-hemolytic streptococci,
  viridans streptococci other than S. pneumoniae
  and S. bovis, Abiotrophia and Granulicatella
  spp., and enterococci E. faecalis and E. faecium)

22
Enterococcus Modes of Infection

• Person-to-person transmission by health care
  providers between patients in the hospital
  setting of Enterococcus faecalis and E. faecium
  (espcially E. faecium) (major mode for hospital
  spread of vancomycin-resistant enterococci)

23
Streptococcus Types of Infectious Disease

• Streptococcus pyogenes is one of the most
  virulent bacterial pathogens, and causes acute
  pharyngitis, impetigo, cellulitis, necrotizing
  fasciitis and myositis (flesh-eating bacteria),
  pneumonia, bacteremia, and streptococcal toxic
  shock syndrome

24
Streptococcus Types of Infectious Disease

• Post-suppurative complications of Streptococcus
  pyogenes include rheumatic carditis (pharyngeal
  infections) due to antibodies against
  streptococcal antigens that cross-react with
  heart tissue and acute glomerulonephritis
  (pharyngeal and skin infections) due to renal
  glomerular deposition of streptococcal antigen -
  antibody immune complexes

25
Streptococcus Types of Infectious Disease

• Neonatal sepsis and meningitis associated with
  maternal infection of the fetus by Streptococcus
  agalactiae during transvaginal delivery
• Streptococcus dysgalactiae causes bacteremia and
  endocarditis
• Streptococcus pneumoniae most common bacterial
  cause of community-acquired pneumonia with and
  without bacteremia

26
(No Transcript)
27
Streptococcus Types of Infectious Disease

• Streptococcus pneumoniae an important cause of
  meningitis, purulent bacterial sinusitis, and
  otitis media

28
Streptococcus Types of Infectious Disease

• Streptococcus bovis bacteremia strongly
  associated with gastrointestinal tract cancer
• Anginosus group of streptococci abscessogenic
  causing occult liver and brain abscess
• Infection by other viridans streptococci occurs
  in neutropenic patients, often due to oral
  mucosal damage caused by chemotherapeutic agents
  or radiation therapy

29
Streptococcus and Endocarditis

• Subacute bacterial endocarditis (especially with
  prosthetic heart valves) due to viridans
  streptococci frequently of the mitis group (S.
  sanguis, S. mitis, S. gordonii, and S. oralis)
• Pyogenic group A, B, C, and G ?-hemolytic
  streptococci
• Streptococcus pneumoniae

30
Enterococcus Types of Infectious Disease

• Urinary tract infection most common usually
  associated with urologic abnormalities
• Intra-abdominal and pelvic infection second most
  common generally polymicrobial with controversial
  etiological role for Enterococcus
• Bacteremia third most common with 2-9
  associated with endocarditis

31
Enterococcus Types of Infectious Disease

• High-level vancomycin resistance results from
  vanA gene coding for abnormal peptidoglycan
  precursors having terminal D-alanyl-D-lactate
  (normal D-alanyl-D-alanine) with decreased
  vancomycin binding affinity and diminshed
  inhibition by vancomycin of cell wall
  peptidoglycan synthesis for VRE strains
  (VREvancomycin-resistant enterococci)
• Associated with resistance to high concentrations
  of vancomycin (vancomycin minimal inhibitory
  concentrations gt 256-µg/ml) and treatment failure
  in VRE infection
• Prevalent with Enterococcus faecium, unusual for
  Enterococcus faecalis

32
Streptococcus Microbiological Properties

• Gram-positive cocci (round to oval shaped,
  occasionally elongated)
• Catalse negative
• Growth on 5 sheep blood agar with optimal growth
  of Streptococcus pneumoniae and occasional
  species of viridans streptococci in the presence
  of 5-10 CO2 (capnophilic)

33
Streptococcus Microbiological Properties

• ?-hemolytic streptococci consist of pyogenic
  large-colony (gt0.5 mm in diameter) forms with
  Lancefield group A, C, or G antigen, and
  small-colony (lt0.5 mm in diameter) forms with
  Lancefield group A, C, F, or G antigen, or no
  Lancefield antigen

34
Streptococcus Microbiological Properties

• ?-hemolytic streptococci are referred to as
  viridans streptococci (viridis, Latin green).
  Non-hemolytic streptococci are considered
  viridans streptococci for preliminary grouping in
  identification
• Viridans streptococci include S. pneumoniae, S.
  bovis, anginosus group, mitis group, mutans
  group, salivarius group, and rare ungrouped
  species

35
Enterococcus Microbiological Properties

• Gram-positive cocci (round to oval-shaped,
  occasionally elongated)
• Catalase negative
• Growth on 5 sheep blood agar with optimal growth
  for occasional strains in the presence of 5-10
  CO2
• ?-hemolysis (E. faecium) and no hemolysis or ?
  hemolysis (E. faecalis)

36
Enterococcus Microbiological Properties

• Positive for hydrolysis of L-pyrrolidonyl-?-naphth
  ylamide (PYR), leucine aminopeptidase (LAP),
  salt-resistant growth (6.5 NaCl), and growth
  resistant to 40 bile with esculin hydrolysis
  (presumptive identification)
• Clinical isolates consist of E. faecalis
  (80-90), E. faecium (5-10), and E.
  casseliflavus, E. avium, E. gallinarum

37
Enterococcus Species Identification (Facklams
Scheme)

• Group I-MAN, SOR, ARG
• E. avium
• Group II-MAN, SOR, ARG
• E. faecalis, E. faecium, E.
• casseliflavus, E. gallinarum
• Group III-MAN, SOR, ARG
• Group IV-MAN, SOR, ARG
• Group V-MAN, SOR, ARG

38
Species Identification of Group II Enterococcus
(Facklams Scheme)1

• MAN SOR ARG ARA PIG
• E. faecalis
  
• E. faecium
  
• E. casseliflavus2
  
• E. gallinarum2
  
• 1MANmannitol, SORsorbose, ARGargininie,
  ARAarabinose, PIGyellow pigment
• 2Motile (E. faecalis and E. faecium non-motile)

39
Enterococcus and Staphylococcus Look Alikes

• Aerococcus viridans
• Gemella
• Stomatococcus

40
Enterococcus and Staphylococcus Look Alikes

• Infection associated with prolonged
  hospitalization, invasive procedures, foreign
  bodies, and antibiotic treatment
• Most frequently recovered in bacteremia and
  endocarditis

41
Enterococcus and Staphylococcus Look Alikes1

• BAP CAT PYR LAP BE NaCl
• Aerococcus2 a /
  /
• Gemella3 a, ß
  /
• Stomatococcus4 ?4 /
  
• 1BAPblood agar plate appearance, CATcatalase,
  PYRpyrrolidonyl-ß-napthylamide, LAPleucine
  aminopeptidase, BEbile-esculin, NaClgrowth in
  6.5 NaCl
• 2A. viridans, staphylococcal-like Gram cocci
• 3Streptococcal-like spherical or elongated Gram
  cocci
• 4Staphylococcal-like Gram cocci with sticky
  colonies

42
Presumptive Identifcation of Streptococcus and
Enterococcus

• Hemolysis sheep blood agar (major)
• PYR test (major)
• CAMP test for synergistic hemolysis
• Bile-esculin hydrolysis test
• Growth in broth containing 6.5 salt
• Optochin susceptibility
• Bile solubilility test
• Voges-Proskauer test

43
Hemolytic Patterns of Growth on Sheep Blood Agar
(Major)

• ?-hemolytic
• Non-?-hemolytic (?-hemolysis or no hemolysis)

44
PYR Test (Major)

• The PYR test is a qualitative procedure that
  determines the ability of an organism to
  hydrolyze L-pyrrolidonyl-
• ?-naphthylamide (PYR) with release of free
  ?-naphthylamide
• Free ?-naphthylamide is detected by reaction with
  dimethylamminocinnam-
• aldehyde which produces a red color

45
(No Transcript)
46
CAMP Test for Synergistic ?-Hemolysis

• The CAMP test was developed by Christie, Atkins,
  and Munch-Peterson whose names provide the
  acronym CAMP. In the CAMP test, group B
  streptococci release a soluble factor (CAMP
  factor) that acts synergistically with the
  ?-hemolysin of Staphylococcus aureus.

47
CAMP Test for Synergistic ?-Hemolysis

• To perform the CAMP test a strain of ?-hemolysin
  producing Staphylococcus aureus is inoculated
  down the center of a sheep blood agar plate. A
  single streak of an isolate to be identified is
  inoculated perpendicular to the S. aureus streak
  to within 3-4 mm of the S. aureus streak. The
  blood agar plate is then incubated overnight in
  an 35-37oC air incubator.

48
CAMP Test for Synergistic ?-Hemolysis

• If a large arrowhead zone of enhanced ?-hemolysis
  develops between the unknown isolate and S.
  aureus the unknown isolate is group B
  Streptococcus.
• Other groups of ?-hemolytic streptococci often
  demonstrate synergism but without the prominent
  arrowhead-shaped zone of enhanced hemolysis.

49
(No Transcript)
50
Bile-Esculin Hydrolysis Test

• The bile-esculin hydrolysis test measures the
  ability of an isolate to hydrolyze the
  ?-glycoside bond of esculin with production of
  esculetin and glucose in the presence of 40
  bile. Free esculetin reacts with the iron salt
  ferric citrate to form a brown to black
  precipitate.

51
Bile-Esculin Hydrolysis Test

• Bile incorporated in agar inhibits growth of most
  streptococci providing selectivity for
  enterococci and group D streptococci, and esculin
  hydrolysis is differential
• Strong bile-esculin reaction with enterococci
  after overnight incubation in ambient air at
  35-37oC, with group D streptococci after 2-3 days
  

52
(No Transcript)
53
Growth in 6.5 NaCl Broth

• Growth of Enterococcus salt-resistant and
  turbidity due to growth apparent after overnight
  incubation in broth containing 6.5 NaCl
• Growth of group D Streptococcus salt-sensitive
  and no turbidity present after overnight
  incubation in 6.5 NaCl-containing broth

54
Growth in 6.5 NaCl Broth

• Resistance or sensitivity of growth to salt
  useful for presumptive identification of
  bile-esculin positive organisms as Enterococcus
  or group D Streptococcus
• PYR test more rapid and has largely replaced
  growth in salt-containing broth

55
Optochin Susceptibility

• The quinine derivative optochin
  (ethylhydroxycupreine hydrochloride) selectively
  inhibits the growth of Streptococcus pneumoniae
• Optochin-containing 6-mm disks placed on sheep
  blood agar cross-streaked with isolate to be
  identified and incubated overnight in a CO2
  incubator

56
Optochin Susceptibility

• Zone of growth inhibition gt 14mm with a 6-mm P
  (optochin) disk indicates inhibition and an
  identification as Streptococcus pneumoniae
• Rare strains of Streptococcus pneumoniae not
  inhibited by a P disk and bile solubility must be
  tested

57
(No Transcript)
58
Bile Solubility Test

• Streptococcus pneumoniae produces autolysins that
  cause central depressions or collapse of
  pneumococcal colonies growing on sheep blood
  agar. Bile salts are surface-active agents that
  enhance autolysis of S. pneumoniae

59
Bile Solubility Tube Test

• Two 0.5 ml aliquots of organism in saline
  (0.5-1.0 McFarland)
• Equivolume of 2 sodium deoxycholate added to one
  aliquot and saline to the second aliquot
• Aliquots incubated at 35oC for 2 hours
• Clearing in the presence of sodium deoxycholate
  positive for bile solubility

60
Bile Solubility Plate Test

• One drop of 10 sodium deoxycholate placed
  directly on sheep blood agar plate colonies of
  organism to be tested
• Plate kept at room temperature or in air
  incubator at 35oC for 15 minutes
• Flattening or disappearance of colonies indicates
  bile solubility

61
Voges-Proskauer Test

• Glucose fermentation requires the reoxidation of
  NADH generated by fermentation back to NAD. This
  is accomplished by the reduction of pyruvic acid
  by NADH to a variety of metabolic products. In
  the butylene glycol pathway this occurs by
  reduction and conden- sation of pyruvic acid to
  acetoin and butylene glycol

62
Voges-Proskauer Test

• Acetoin and butylene glycol are detected in the
  Voges-Proskauer test by oxidation to diacetyl at
  an alkaline pH and addition of ?-naphthol that
  forms a pink-red product within 5-15 minutes

63
Voges-Proskauer Test

• Positive Voges-Proskauer test with minute-colony
  (lt0.5 mm) group A, C, F, or G or non-groupable
  ?-hemolytic streptococci, or optochin-resistant,
  bile-esculin negative viridans streptococci
  indicates anginosus group of Streptococcus
• Acetoin results in a distinctive butter-scotch
  odor with blood agar cultures

64
Algorithm for facultative catalase-negative
gram-positive cocci
65
Algorithm ?-Hemolytic, PYR

• Streptococcus pyogenes Colonies gt 0.5 mm (SBAP),
  Lancefield group A antigen
• Enterococcus faecalis Bile-esculin positive

66
Algorithm ?-Hemolytic, PYR

• Streptococcus agalactiae CAMP, Lancefield group
  B antigen
• Streptococcus dysgalactiae subspecies equisimilis
  Colonies gt 0.5 mm (SBAP), Lancefield group C or G
  antigen
• Anginosus group of Streptococcus Colonies lt 0.5
  mm (SBAP), VP, Lancefield group A, C, F, or G
  antigen, or non-groupable by Lancefield antigen

67
Algorithm Non-?-Hemolytic, PYR

• Enterococcus salt resistance bile-esculin
• Abiotrophia, Granulicatella Pyridoxal-dependent
  growth (nutritional variant streptococci)

68
Algorithm Non-?-Hemolytic, PYR

• Streptococcus pneumoniae Optochin sensitive, bile
  soluble
• Streptococcus bovis bile esculin
• Anginosus group of Streptococcus VP
• Leuconostoc, Pediococcus High-level vancomycin
  resistance

69
(No Transcript)
70
Six Species Groups of Streptococcus Based on 16S
rRNA Gene Homology

• Pyogenic beta-hemolytic large-colony
• forming group
• Non-pyogenic groups (viridans streptococci)
• Anginosus
• Bovis
• Salivarius
• Mutans
• Mitis

71
Grouping of the viridans streptococci

• Primary Grouping
• Voges-Proskauer (acetoin production)
• Arginine hydrolysis
• Secondary Grouping
• Esculin hydrolysis
• Lancefield group D
• Melibiose fermentation
• Sorbitol fermentation
• Urea hydrolysis

72
Primary grouping of the viridans streptococci

• VP ARG
• Anginosus group
• VP ARG
• Bovis group
• Salivarius group (other than S. vestibularis)
• Mutans group
• VP ARG or
• Mitis
• Salivarius (S. vestibularis)

73
Secondary grouping of the viridans streptococci
(VP ARG)

• Esculin D-Ag Melibiose
  Sorbitol
• Bovis1,2
• Mutans /
  
• Salivarius
  
• 1For group D-negative strains, S. bovis
  melibiose and sorbitol
• 2S. bovis bile esculin , PYR with no growth in
  6.5 NaCl or at 45oC

74
Secondary grouping of the viridans streptococci
(VP ARG or )

• Urea
• Salivarius group (S. vestibularis)
• Urea
• Mitis group

75
Recommended Reading

• Winn, W., Jr., Allen, S., Janda, W., Koneman,
• E., Procop, G., Schreckenberger, P., Woods,
• G.
• Konemans Color Atlas and Textbook of
• Diagnostic Microbiology, Sixth Edition,
• Lippincott Williams Wilkins, 2006
• Chapter 13. Streptococci, Enterococci, and the
  Streptococcus Like Bacteria

76
Recommended Reading

• Murray, P., Baron, E., Jorgensen, J., Landry,
• M., Pfaller, M.
• Manual of Clinical Microbiology, 9th
• Edition, ASM Press, 2007
• Spellerberg, B., and Brandt, C. Chapter 29.
  Streptococcus
• Teixeira, L.M., Carvalho, M. Da G.S., and
  Facklam, R.R. Chapter 30. Enterococcus.
• Ruoff, K.L. Chapter 31. Aerococcus,
  Abiotrophia, and Other Aerobic Catalase-Negative,
  Gram-Positive Cocci