teaching support for 2nd year medical school students: steps of the laboratory diagnosis of infections caused by bacteria of the genera Staphylococcus and Streptococcus

1. Bacteriological diagnosis of infections
caused by bacteria of
Staphylococcus and Streptococcus genera
- Part Two -
http://www.slideshare.net/DanaSinzianaBreharCi/
staphylococcus-streptococcus-bacteriological-diagnosisii-45259850

2. Gram positive cocci
• Family:
Micrococaceae
• Genera:
– Staphylococcus
– Micrococcus
– Stomatococcus
– Planococcus
• Family:
Streptococacceae
• Genera:
– Streptococcus
– Enterococcus
– Aerococcus
– Gemella
– Leuconostoc
– Pediococcus
– Lactococcus

3. Classification of streptococci
Criteria:
• I. Type of hemolysis produced by bacterial growth on
blood agar
• II. Antigenic structure (Lancefield)

4. Classification of streptococci according to
type of hemolysis
• β-hemolytic streptococci:
– Complete, clear hemolysis (medium around the colony is
transparent = bacterial growth produced complete digestion of
red blood cells in the blood agar) e.g. Streptococcus pyogenes
• α-hemolytic streptococci:
– partial hemolysis (medium around the colony is translucent and
greenish = bacterial growth produced incomplete digestion of
hemoglobin in the blood agar (conversion of hemoglobin to
methemoglobin) e.g. Streptococcus viridans, Streptococcus
pneumoniae)
• Non-hemolytic streptococci

5. Blood agar:
Enterococcus fecalis (non-hemolytic/variable) and
Streptococcus pyogenes (β-hemolysis)

6. Streptococcus pyogenes on blood agar

7. Left: Alpha hemolysis – Streptococcus pneumoniae
Right: Beta hemolysis – Streptococcus pyogenes

8. Colonies of α-hemolytic streptococci

9. Classification of streptococci according to
antigenic structure (Lancefield grouping)
Rebecca Lancefield (1895-1981)
(American microbiologist at the
Rockefeller Institute for Medical
Research)
• based upon the C polysacharidic antigen (group-specific) in
bacterial wall → groups A – H and K-V
• based upon M and T proteins (type specific) → over 80 types of
group A streptococci
• !! Lancefield grouping does not include streptococci lacking group
antigens e.g. Str.pneumoniae, Str.viridans, etc.)

11. Classification of streptococci

12. Genus Streptococcus
• Clinically significant microbial species:
– Streptococcus pyogenes (group A): cellulitis, pharyngitis,
scarlet fever + complications: articular (acute rheumatic fever),
cardiac (rheumatic carditis), renal (glomerulonephritis)
– Streptococcus agalactiae (group B): severe infections in
neonates (pneumonia, meningitis), urogenital infections, sepsis
– Streptococcus pneumoniae: pneumonia, bronchopneumonia,
meningitis
– Viridans streptococci: Streptococcus mutans, Streptococcus
sanguis, Streptococcus anginosus (dental caries, periodontal
disease + septicaemia, endocarditis)

13. Streptococcus pyogenes
(group A)
- clinical significance -
• Acute, respiratory infections: pharyngitis, scarlet fever +
complications: articular (acute rheumatic fever), cardiac
(rheumatic carditis), renal (glomerulonephritis)
• Skin infections: erysipelas, impetigo, intertrigo, pemfigus,
celulitis, abscesses + complications: sepsis

14. Streptococcus pyogenes:
Steps of bacteriological diagnosis
• Collection of specimens (e.g. pus, pharyngeal exudate,
content of vesicles, CSF, urine, etc)
• Microscopic examination
• Inoculation of culture media
• Biochemical tests
• Agglutination tests
• Serological diagnosis
• Antimicrobial susceptibility tests (antibiogram)

15. ”Strep throat” – Pharyngitis with Streptococcus pyogenes:
left – petechiae; right – pus deposits

16. Scarlet fever:
red rash; ”strawberry tongue”

17. Erysipelas – streptococcal infection of the
dermis and superficial lymph vessels

18. Impetigo – non-bulous and bulous

19. Streptococcus pyogenes
– Microscopic examination -
Gram stained smears:
• Cocci:
– Round / ovoid shape; arranged in chains /
pairs
– Gram positive
– Aerobic growth (+anaerobic)

20. Streptococcus pyogenes: Gram stained smear:
ovoid Gram positive cocci, arranged in chains

21. Streptococcus – Gram stained smear
• Chains of ovoid, Gram
positive cocci

22. Streptococcus – Gram stained smear

23. Streptococcus pyogenes
- Cultivation & isolation -
• Blood containing media e.g. blood agar, Todd-Hewit
broth, SSP (selective medium for streptococci and
pneumococci)
• Most frequently:
– (Initial inoculation of selective medium (Pick) – favours growth
and multiplication of streptococci and inhibits other bacterial
species)
– ↓
– Reinoculation on 5% sheep blood agar

24. Streptococcus pyogenes
- identification -
• Colonial characters:
– small, pinpont, 0.5 µM diameter, transparent
– β-hemolysis - complete digestion of red blood cell contents
surrounding colony
• Group identification:
1. bacitracin sensitivity test – group A streptococci are bacitracin
sensitive / other streptococci are resistant
2. Immune-based tests (Ag-Ab) – agglutination/precipitation

25. Streptococcus pyogenes on blood agar
• Small, pinpoint
colonies
• Complete hemolysis
(transparent area around
colonies)

26. Streptococcal group identification
1. Bacitracin sensitivity test
• used to determine the effect of
a small amount of bacitracin
(0.04 U) on an organism.
• Streptococcus pyogenes
(group A) is inhibited
(minimum 10 mm inhibition
diameter) by the small amount
of bacitracin in the disk; other
beta-hemolytic streptococci
usually are not

27. Streptococcal group identification
2. Ag-Ac reactions
• Principle: contact between streptococcal group Ag (C
polysacharide) and homologous Ab (serum anti-group
A / B / C...= ”antisera”)
• the C polysacharide is located within the bacterial wall
• ↓
• Preliminary extraction is required

28. Streptococcal group identification
2. Ag-Ac reactions - Methods
1. ”Lancefield” precipitation
• extraction of the C polysacharide with HCl + heating
• contact with set of antisera (anti-group A/B...)
1. Agglutination
• enzymatic extraction of the C polysacharide
• contact with set of antisera (anti-group A/B...)

29. Streptococcal group identification
2. Ag-Ac reactions – Methods - continued
3. Co-agglutination
• enzymatic extraction of the C polysacharide
• slide agglutination with antisera adsorbed on protein A of
staphylococci
4. Latex agglutination
- Similar with co-agglutination; group specific Ab (antisera)
adsorbed on latex particles

30. Streptococcal type identification
Performed for epidemiologic purposes
• M typing:
– precipitation in capillary tubes with anti-M sera
– typing based upon M protein (virulence factor with anti-
phagocytic capacity)
• T typing:
– slide agglutination with anti-T sera
• PCR – for non-typable germs

31. Antimicrobial sensitivity
• Streptococcus pyogenes – sensitive to penicillin (no
antibiogram required);
• Final step of bacteriological diagnosis: perform the
bacitracin sensitivity test on a culture of β-hemolytic
streptococci – POSITIVE test = group A

32. Streptococcus pyogenes
- Serological diagnosis -
• Used in case of late complications – germs are difficult to
detect in culture
• Detection of serum antibodies against bacterial:
– products:
• streptolysin O (toxin responsible for hemolysis),
• streptokynase (enzyme responsible for fibrinolysis – clot lysis)
– components:
• M antigen, MAP (M antigen associated protein)

33. Streptococcus pyogenes
- Serological diagnosis - continued
• ASLO test = detection of serum anti-streptolysin O
antibodies in patient serum
• SLO present in streptococci of group A + C and G
• Principle: ASLO Ab neutralize the effect of streptolysin O
(SLO) on red blood cells (= annihilation of hemolysis)

34. ASLO test - Reaction steps:
• tubes with serial dilutions of patient serum
• add standard amount of streptolysin O
• incubation
– IF ASLO Ab present in patient serum → SLO blocked in Ag-Ab
complex – will not act upon red blood cells (see below)
– IF ASLO Ab absent in patient serum → no Ag-Ab complex →
SLO not neutralized – will act upon red blood cells (see below)
• add suspension of rabbit red blood cells (RBC)
– POSITIVE result = no hemolysis (SLO neutralized by Ab in
patient serum); RBC deposit at the bottom of tube
– NEGATIVE result = hemolysis (no Ab to neutralize SLO; SLO
acts upon red blood cells → hemolysis)

35. ASLO test – Reading the results
• The titre of ASLO Ab = the dilution in the last tube where
hemolysis is absent
• Normal titer values: 166-200 u/ml
• ASLO titers 200 u/ml = Recent infection˃
• repeated testing e.g. after angina with Streptococcus
group A: increasing titer up to a maximum ~2500 u/ml in
5 weeks; decrease to normal values within 6-12 months
= cured infection

36. ASLO-latex:
rapid detection of ASLO Ab by slide agglutination

37. Streptococcus agalactiae (group B)
- clinical significance -
• Comensal: intestine, vagina, male urethra, upper airways
• Clinical significance: Severe infections:
– in newborns:
• pneumonia acquired during birth (maternal vaginal colonization
/infection)
• meninigitis – hospital acquired infection
– In immune-compromised adults/elderly:
• Meningitis, myocarditis, respiratory infections, sepsis

38. Streptococcus agalactiae (group B)
Steps of the bacteriological diagnosis
• Collection of specimens:
– Vaginal / cervical secretion, CSF
• Microscopic examination:
– Gram positive cocci, chains/pairs/isolated – no particular
diagnostic value
• Innoculation of culture media:
– blood agar, “chocolate” agar
– Colonial characters:
• colonies – slightly larger than group A streptococci,
• hemolysis – variable / partial / “foggy” (α´-hemolysis)
Not enough! Further tests required…

39. Streptococcus agalactiae:
Gram positive ovoid cocci in short chains

40. Streptococcus agalactiae (group B)
The CAMP* test
Principle:
• hemolysis produced by Staphylococcus aureus acts synergistically
with the CAMP factor produced by Streptococcus agalactiae (group
B)→ enhanced hemolysis in the region where the 2 cultures meet
• CAMP factor – pathogenicity factor - extracellular cytolysin (cytolytic
protein) that binds the Fc fragments of immunoglobulin G (IgG),
similar to protein A of Staphylococcus aureus
• Protocol: co-cultivation of S.aureus and (suspected) Streptococcus
agalactiae (group B); plate inoculation by PERPENDICULAR,
SINGLE STREAKS
*acronym for "Christie–Atkins–Munch-Petersen",for the three
researchers who discovered the phenomenon

41. The CAMP test - continued
(A) Streptococcus (group
B) positive test
(enhanced hemolysis)
(B) Streptococcus
pyogenes (group A)
negative test
(C) Staphylococcus
aureus

42. Streptococcus agalactiae (group B)
Steps of the bacteriological diagnosis -
continued
• Antimicrobial susceptibility testing – mandatory!
• WHY?: Streptococcus agalactiae strains resistant to
penicillin have been isolated

43. Classification of streptococci

44. Viridans streptococci
• Group of streptococci which are comensal in:
– oral cavity (a.k.a. oral streptococci)
– Intestine
– Skin
– Genital tract
• Oral streptococci: Streptococcus mutans, Streptococcus
sanguis, Streptococcus anginosus (dental caries,
periodontal disease + septicaemia, endocarditis)
• No Lancefield antigens !!

45. Viridans streptococci - continued
• Sreptococcus mutans - etiologic agent of dental
caries
• Other streptococci may be involved in other mouth or
gingival infections
• If introduced into the bloodstream (e.g. following tooth
extraction) - potential of causing endocarditis,
particularly in individuals with damaged heart valves (the
most common causes of subacute bacterial
endocarditis)

46. Viridans streptococci:
α hemolysis on blood agar

47. Classification of streptococci

48. Streptococcus pneumoniae
• Clinical significance:
• comensal germ of the upper airways; may become
pathogenic when encapsulated
• Meningitis, pneumonia, broncho-pneumonia

49. Streptococcus pneumoniae
Steps of bacteriological diagnosis
• Collection of specimens: sputum, CSF, blood, etc
• Macroscopic examination: “rusty” sputum
• Microscopic examination:
– Gram stained smears: white blood cells + Gram positive cocci, in
diplo/short chains; characteristic aspect: two candle flames ◄►
touching with their basis
• Cultivation:
– blood agar (in CO2 atmosphere), 37°C, 24 hrs;
– colonial characters: small, smooth colonies with α-hemolysis
(green); colonies become depressed in time (autolysis) =
differentiation from viridans streptococci

51. Streptococcus pneumoniae – Gram stained
sputum smear

52. Streptococcus pneumoniae on blood agar

53. Left: Alpha hemolysis – Streptococcus pneumoniae
Right: Beta hemolysis – Streptococcus pyogenes

54. Alpha-hemolysis: Streptococcus
pneumoniae

55. How to differentiate between
α-hemolytic streptococci?
• Streptococcus pneumoniae ≠ Viridans streptococci
• Autolysis: self digestion of colonies in time (present in
Str.pneumoniae and absent in viridans str.) – see above
• Bile solubility test
• Optochin sensitivity test

56. The Bile solubility test
• Principle: Bile salts selectively lyse Streptococcus
pneumoniae when added onto cultures on agar / broth
• POSITIVE TEST on agar: colony ”disappears” upon
addition of bile salts
• POSITIVE TEST in broth: turbidity of broth (reflecting
bacterial growth) clarifies upon addition of bile salts
• Negative test: Streptococcus viridans (bile-insoluble)

57. The Bile solubility test

58. Streptococcus pneumoniae
The optochin sensitivity test
• Streptococcus pneumoniae
(optochin sensitive) – bacterial
growth inhibited around the
optochin impregnated disk
• viridans streptococci (optochin
resistant)

59. Gram positive cocci
• Family:
Micrococaceae
• Genera:
– Staphylococcus
– Micrococcus
– Stomatococcus
– Planococcus
• Family:
Streptococacceae
• Genera:
– Streptococcus
– Enterococcus
– Aerococcus
– Gemella
– Leuconostoc
– Pediococcus
– Lactococcus

60. Classification of streptococci

61. Genus Entreococcus
• Previously classified as group D Streptococcus
• Clinical significance:
– endocarditis, uro-genital infections, meningitis
• Steps of bacteriological diagnosis:
– Collection of specimens – depending on infection site
– Microscopy: Gram positive cocci, short chains/pairs
– Cultivation: blood agar – small nonhemolytic colonies;
– identification:
• group D polysacharide
• biochemical tests

62. Blood agar: Enterococcus fecalis (non-hemolytic)
and Streptococcus pyogenes (hemolytic)

63. Enterococcus fecalis on blood agar

64. Vancomycin Resistant Enterococcus fecalis (VRE)
on selective medium containing vancomycin

65. To sum up:
• logical steps of the bacteriological diagnosis
↓
• Id. of Staphylococcus / Streptococcus infections
(similarities & differences)

66. Microscopy: Gram stained smears
Similar shape; Different arrangement

67. Staphylococcus / Streptococcus (?)

68. Cultivation (blood agar) –
absence / presence & type of hemolysis
Staphylococcus Streptococcus

69. Blood agar plates
Left: Staphylococcus; Right: Streptococcus

70. Cultivation on selective media
• Chapman agar (mannitol salt):
– high salt content – inhibits Streptococcus, allows
Staphylococcus growth
– Mannitol fermentation – differentiates between:
• Staphylococcus aureus (manitol fermentation – changes the colour
of medium from pink to yellow) and
• Staphylococcus epidermidis (no manitol fermentation – medium
colour remains pink)

71. Biochemical differentiation
staphylococci / streptococci
CATALASE TEST
• Principle: catalase decomposes hydrogen peroxide into
water and oxygen (gas bubbles)
• 2-3 drops of hydrogen peroxide placed on a colony
• POSITIVE TEST: rapid effervescence
• Staphylococcus - POSITIVE / Streptococcus - Negative
__________
Catalase (+) = Staphylococcus ...
→ continue with further identification

72. Differentiation between staphylococci i.e.
after positive catalase test
COAGULASE TEST
Principle: the coagulase of Staphylococcus
aureus (aka ”clumping factor”) converts
fibrinogen into fibrin →clot
Staphylococcus aureus – POSITIVE TEST
Other staphylococci (e.g. Staphylococcus
epidermidis, Staphylococcus
saprophyticus) – Negative test

73. Differentiation between staphylococci
Staphylococcus
aureus
Staphylococcus
epidermidis
Staphylococcus
saprophyticus
Catalase + + +
Coagulase + - -
Colonies on
blood agar
Golden yellow white White/yellowish
Hemolysis Complete (β) none None
Mannitol
fermentation
+ - -

74. Biochemical differentiation
staphylococci / streptococci
CATALASE TEST
• Principle: catalase decomposes hydrogen peroxide into
water and oxygen (gas bubbles)
• 2-3 drops of hydrogen peroxide placed on a colony
• POSITIVE TEST: rapid effervescence
• Staphylococcus - POSITIVE / Streptococcus - Negative
__________
Catalase (-) = Streptococcus ...
→ continue with further identification

75. Differentiation between streptococci i.e. after
negative catalase test
Type of hemolysis Lancefield group
• A = Streptococcus
pyogenes
• B = Streptococcus
agalactiae
• D = Enterococcus
Nongroupable
• Viridans streptococci
• Streptococcus
pneumoniae

76. Differentiation between streptococci i.e. after
negative catalase test - continued
BACITRACIN SENSITIVITY TEST
• (+) Streptococcus pyogenes / (-) Streptococcus
agalactiae
BILE SOLUBILITY TEST
• (+) Streptococcus pneumoniae / (-) Viridans streptococci
OPTOCHIN SENSITIVITY TEST
• (+) Streptococcus pneumoniae / (-) Viridans streptococci

77. Differentiation between streptococci
Lancefield
group
Hemolysis Other dg characters
Streptococcus
pyogenes
A β Bacitracin sensitive;
CAMP test (-)
Streptococcus
agalactiae
B β Bacitracin resistant;
CAMP test (+)
Enterococcus D None /
variable
Streptococcus
pneumoniae
Not
applicable
α Bile soluble;
Optochin sensitive
Viridans
streptococci
Not
applicable
α Not bile soluble;
Optochin resistant

78. Staph. aureus - mannitol fermentation (left side, left plate)
Staph.epidermidis - no mannitol fermentation (right side, left plate)
Streptococcus – plate on the right