MLAB 2434: Microbiology Keri Brophy-Martinez

1
MLAB 2434 MicrobiologyKeri Brophy-Martinez

• Streptococci, Enterococci and Other
  Catalase-Negative Gram Positive Cocci

2
Streptococcus and Enterococcus General
Characteristics

• Members of the Streptococcaceae family
• Facultatively anaerobic
• Aerotolerant
• Catalase negative

3
Streptococcus and Enterococcus General
Characteristics

• Most are typically spherical some may appear
  elongated or ovoid
• They may appear in chains or pairs

4
Streptococcus and Enterococcus Habitat and
Clinical Infections

• Clinical Infections
• Upper and lower respiratory tract infections
• Urinary tract infections
• Wound infections
• Endocarditis

• Habitat
• Normal Flora
• Respiratory tract
• Gastrointestinal tract
• Urogenital tracts

5
Streptococcus and EnterococcusCell Wall
Structure

• Thick peptidoglycan layer
• Teichoic acid
• Carbohydrate layer present
• Used in Lancefield grouping of Streptococcus spp.
• Capsule
• Virulence factor
• S. pneumoniae

6
Classification Overview

• Physiologic characteristics
• Pyogenic produce pus
• Lactococci found in dairy products
• Enterococci normal gut flora
• Viridans normal URT flora
• Hemolysis
• J. H Brown
• Alpha, beta, gamma classifications
• Serological grouping
• Typing of C carbohydrate
• Lancefield group
• Performed only on ß-hemolytic hemolysis
• Biochemical
• Based on reaction of isolate

7
ClassificationHemolysis

• J.H. Brown- 1903
• Grouped streps on ability to lyse RBCS
• Alpha
• Beta
• Gamma
• Alpha-prime

8
Hemolysis Patterns

• Alpha (a)
• Greenish discoloration
• Caused by partial lysis of RBCs in media

9
Hemolysis Patterns

• Beta (ß)
• Complete lysis of RBCs
• Produces a clear, colorless zone

10
Hemolysis Patterns

• Gamma
• Colonies show no hemolysis or discoloration
• Called non-hemolytic

11
ClassificationSerological Grouping

• Rebecca Lancefield 1930
• Based on presence of carbohydrates in cell wall
• Groups A, B, C, and D most significant
• Typing done on beta-hemolytic colonies

12
ClassificationBiochemical Identification/Suscep
tibility

• Bacitracin
• A disk or Taxo A disk
• 0.04 units
• Identifies Group A streptococci (S. pyogenes)
• Zone of inhibition is presumptive ID of Grp. A
  strep

Group A streptococcus is susceptible to A disk
(left)
13
Biochemical Identification/Susceptibility

• Optochin
• P disk orTaxo P disk
• Differentiates S. pneumoniae from other
• alpha-hemolytic streptococci

14
Biochemical Identification

• Bile solubility test
• Detects amidase enzyme
• Under bile salt or detergent lyses cell wall
• Clearing through lysis of colonies
• Diagnostic for S. pneumoniae

15
Biochemical Identification

• PYR hydrolysis
• Substrate L-pyrrolidonyl-b-napthlyamide (PYR) is
  hydrolyzed by the enzyme pyrrolidonyl arlamide
• Group A Streptococci and Enterococcus sp. posses
  the necessary enzyme.
• More specific than Bacitracin for Group A
  streptococci

The disk on the right has turned a red color,
indicating a indicating a positive reaction. The
left disk remains a yellow color indicating a
negative result.
16
Biochemical Identification

• Hippurate hydrolysis
• Differentiates Group B streptococci from other
  beta hemolytic streptococci
• Group B streptococci hydrolyzes sodium hippurate
  forming a purple color

17
Biochemical Identification

• CAMP test
• Christie,Atkins, Munch-Petersen
• Detects the production of enhanced hemolysis that
  occurs when b-lysin and the hemolysins of Group B
  streptococci come in contact with each other

Group B streptococci showing the classical
arrow-shaped hemolysis near the staphylococcus
streak
18
Biochemical Identification

• Bile esculin hydrolysis
• Ability to grow in bile and hydrolyze Esculin
• Characteristic of streptococci that possess
  group D antigen and Enterococci

Both Group D streptococci and enterococci produce
a positive (top) bile Esculin hydrolysis test.
19
Biochemical Identification

• Salt Tolerance
• Growth in 6.5 NaCl broth
• Differentiates Group D streptococci from
  enterococci
• Enterococcus POSITIVE
• Tube on left
• Group D Streptococcus NEGATIVE
• Tube on right

20
Non-culture Identification

• Slide agglutination kits
• Latex beads are coated with group specific
  anti-serum, which clump when mixed with a small
  amount of colony from the specific Streptococcus
  sp.
• Nucleic Acid Probes
• Detect genes for specific groups

21
Slide Agglutination Tests
22
Slide Agglutination Tests
23
Break Time!
24
Virulence Factors Streptococcus pyogenes

• Fimbrae Protein F
• Attachment and adherence
• M protein
• Resistance to phagocytosis
• Hyaluronic acid capsule
• Prevents phagocytosis
• Lipoteichoic acid
• Adheres to molecules on host epithelial cells

25
Virulence FactorsStreptococcus pyogenes

• Hemolysins
• Streptolysin O (O2 labile) detected in ASO titers
• Streptolysin S (O2 stable) Causes hemolysis on
  plates
• Erythrogenic toxin/Streptococcal pyogenic
  exotoxin
• Scarlet fever
• Enzymes
• Streptokinase
• DNases
• Hyaluronidase spreading factor

26
Clinical Conditions Streptococcus
pyogenes(Group A)
27
Clinical Conditions Streptococcus
pyogenes(Group A)

• Pyodermal infections
• Impetigo weeping lesion
• Erysipelas
• Cellulitis
• Wound Infections

Erysipelas due to Streptococcus pyogenes
28
Clinical Conditions Streptococcus
pyogenes(Group A)

• Scarlet Fever
• Starts with pharyngitis and causes rash on trunk
  and extremities
• Due to untreated Group A infections

29
Invasive Group A Streptococcal Infections

• Streptococcal toxic shock syndrome
• Multi-organ system failure similar to
  staphylococcal toxic shock
• Initial infection may have been pharyngitis,
  cellulitis, peritonitis, or other wound infections

30
Invasive Group A Streptococcal Infections

• Cellulitis/Necrotizing Fasciitis
• Severe form of infection that is life-threatening
• Bacteremia and sepsis may occur
• In patients necrotizing fasciitis, edema,
  erythema, and pain in the affected area may
  develop
• Streptococcal myositis resembles clostridial
  gangrene

31
PostGroup-A Streptococcal Infections

• Rheumatic fever
• Fever
• Inflammation of the heart, joints, blood vessels,
  and subcutaneous tissues
• Chronic, progressive damage to the heart valves
  (most evidence favors cross-reactivity between
  Strep. antigens and heart tissue)
• ASO titer will be elevated

32
PostGroup-A Streptococcal Infections

• Acute glomerulonephritis (AGN)
• Follows either cutaneous or pharyngeal infections
• More common in children than adults
• Antigen-antibody complexes deposit in the
  glomerulus
• Inflammatory response causes damage to the
  glomerulus and impairs the kidneys

33
Laboratory Diagnosis Group A Streptococcus
Grams stained wound smear showing gram-positive
cocci in chains with numerous polys (PMNs)
34
Laboratory Diagnosis Group A Streptococcus

• Colony morphology
• Transparent, smooth, and well-defined zone of
  complete or b- hemolysis

35
Laboratory Diagnosis Group A Streptococcus

• Identification
• Catalase-negative
• Bacitracin-susceptible
• PYR-positive
• Hippurate hydrolysis- negative
• Slide agglutination

Group A streptococci is susceptible to Bacitracin
disk (left) The right shows resistance
36
Group B b-Hemolytic Streptococcus (Streptococcus
agalactiae)

• Colonize the urogenital tract of pregnant women
  (10-30 rate can cause OB complications such as
  premature rupture of membranes and premature
  delivery)
• Mother fails to pass protective antibodies to
  fetus
• Cause invasive diseases in newborns
• Early-onset infection
• Late-onset disease

37
Invasive Disease in the Newborn
Early Onset Late-Onset
Age of Onset lt 7 days 7 30 days
Median age of onset 1 hour 27 days
Maternal complications of labor Common Less common
Incidence of prematurity 25 Less common
Source of Organism Maternal genital tract Maternal genital tract nosocomial community
Clinical presentation Nonspecific (35-55 ) Meningitis 5-10 Respiratory diseases 35-55 Focal Meningitis 25-35
Types I, II III, V III (75)
Mortality Rate 5-15 2-10
38
Invasive Streptococcus agalactiae Infections

• In adults
• Occurs in immunosuppressed patients or those with
  underlying diseases
• Often found in a previously healthy adult who
  just experienced childbirth

39
Laboratory Diagnosis Streptococcus agalactiae

• Colony morphology
• Small
• Grayish-white
• Mucoid, creamy
• Narrow zone of b-hemolysis

40
(No Transcript)
41
Laboratory Diagnosis Streptococcus agalactiae

• Presumptive Identification tests
• Gram stain- GPC in chains
• Catalase-negative
• Bacitracin-resistant
• Bile esculin- negative
• Does not grow well in 6.5 NaCl.
• CAMP- positive
• Slide agglutination

S. agalactiae shows the arrow-shaped hemolysis
near the staphylococcus streak, showing a
positive test for CAMP factor
42
Streptococcus pneumoniae

• General characteristics
• Inhabits the nasopharyngeal areas of healthy
  individuals
• Typical opportunist
• Possess C substance
• Virulence factors
• Polysaccharide capsule

43
Clinical ConditionsStreptococcus pneumoniae

• Pneumonia
• Most common cause of bacterial pneumonia
• Meningitis
• Bacteremia
• Sinusitis/otitis media
• Most common cause of otitis media in children lt 3
  years

44
Laboratory DiagnosisStreptococcus pneumoniae

• Microscopic morphology
• Gram-positive cocci in pairs lancet-shaped
  (somewhat oval in shape)

45
Laboratory DiagnosisStreptococcus pneumoniae

• Colony morphology
• Smooth, glistening, wet-looking, mucoid
• a-Hemolytic
• CO2enhances growth
• As colony ages, autolytic collapse causes
  checker shape

46
Laboratory Diagnosis Streptococcus pneumoniae

• Identification
• Catalase negative
• Optochin-susceptibility-testsusceptible
• Bile-solubility-testpositive

47
Identification Schema
48
Enterococcus Species

• Clinically Significant Isolates
• E. faecalis
• E. faecium
• Opportunistic pathogens
• In the GI tract, genitourinary tract and oral
  cavity
• Associated infections
• Bacteremia
• Urinary tract infections
• Wound infections
• Endocarditis
• Hospital-acquired Infections

49
Laboratory Diagnosis Enterococcus Species

• Microscopic morphology
• Cells tend to elongate
• Colony morphology
• Small, grey
• Most are non-hemolytic, although some may show a-
  or, rarely, b-hemolysis
• Possess Group D antigen

50
Laboratory Diagnosis Enterococcus Species

• Identification tests
• Catalase may produce a weak catalase reaction
• Hydrolyze bile esculin
• Differentiate Group D from Enterococcus sp. with
  6.5 NaCl or PYR test
• Important to identify Enterococcus from
  non-Enterococcus, because Enterococcus must be
  treated more aggressively.

51
Identification Schema
Or PYR disk
52
Other Streptococcal Species

• Viridans group (Viridans means green)
• Members of the normal oral, nasopharyngeal flora,
  GI tract and female genital tract
• Most are ? hemolytic but also includes
  nonhemolytic species
• The most common cause of subacute bacterial
  endocarditis (SBE)
• Also involved with gingivitis and dental carries
• PYR negative
• Optochin negative
• Bile solubility negative

53
Viridans

• 5 groups
• Anginosus
• S. anginosus, S. intermedius, S. constellatus
• Mitis
• S. sanguig, S. parasanguis, S. gordonii, S.
  crista, S. infantis, S. mitis, S. oralis, S.
  oralis, S. peroris
• Mutans
• S. criceti, S. downei, S. macacae, S. mutans, S.
  rattus, S. sobrinus
• Salivarius
• S. salivarius, S. thermophilus, S. vestibularis
• Bovis
• S. equinus, S. gallolyticus,S. infantarius, S.
  alactolyticus

54
Abiotrophia Granulicatella

• Once referred to as Nutritionally variant
  streptococci (NVS)
• Causes endocarditis and otitis media
• Normal flora of oral cavity
• Requires pyridoxal to grow (can satellite around
  Staph, E. coli, Klebsiella, Enterobacter and
  yeasts)

55
Streptococcus and Enterococcus
56
Streptococcus and Enterococcus

• Treatment
• Generally, streps are not routinely tested for
  susceptibility since penicillin drug of choice.
  If the patient is allergic to pen use
  erythromycin.
• Antibiotic resistance seen with Enterococcus, use
  vancomycin

57
References

• http//archive.microbelibrary.org/ASMOnly/Details.
  asp?ID2566
• http//www.goodtoknow.co.uk/health/Scarlet-fever
• http//onwardstate.com/2009/12/10/keep-your-goals-
  to-yourself/
• Mahon, C. R., Lehman, D. C., Manuselis, G.
  (2011). Textbook of Diagnostic Microbiology (4th
  ed.). Maryland Heights, MO Saunders.