The document outlines the bacteriological diagnosis of infections caused by specific genera of bacteria, mainly Staphylococcus and Streptococcus. It details the classification of streptococci based on hemolysis and antigenic structure, emphasizing clinically significant species like Streptococcus pyogenes and Streptococcus pneumoniae, along with their respective diagnosis methods. Specific diagnostic steps, including specimen collection, culture inoculation, and identification tests, are also described for effective determination of infections.

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