1. Laboratory Diagnosis
• Colonial Morphology (BAP)
• S. aureus  Golden yellow colonies, smooth, entirely raised, -
hemolytic (>24 hrs of incubation)
• S. epidermidis  translucent, gray-white colonies, non-
hemolytic
• S. saprophyticus  (same as S. epidermidis)

2. Laboratory Diagnosis
Gram stain: Gram-positive cocci
Differentiates
Staphylococc
us and Catalase test
Streptococcu
s
Staphylococcus Streptococcus
sp. sp.

3. Catalase test (Slide)
Negative Positive

4. Medically important Staphylococci
• Staphylococcus aureus
• Staphylococcus epidermidis
• Staphylococcus saprophyticus

5. Laboratory Diagnosis
Catalase test Coagulase test/
MSA
Staphylococcus
sp. S. aureus
S. saprophyticus
S. epidermidis

6. Coagulase Test
• 2 types
1.) Bound/Clumping factor
 Slide test
 (+) Result: agglutination of organism when mixed w/
plasma
 not all strains of S. aureus produced clumping factor
2.) Free
 Tube test
 (+) Clot formation
Anticoagulant: EDTA

7. Coagulase test (Tube)
Positive Negative

8. Mannitol Salt Agar
• pH indicator:
phenol red
• Sugar:
Mannitol
• Salt conc.:
10%
• (+) Result:
Yellow Halo

9. Laboratory Diagnosis
Coagulase test/
Novobiocin test
MSA
(S) (R)
S. saprophyticus
S. epidermidis
S. epidermidis
S. saprophyticus

10. Novobiocin disc test
S. epidermidis S. saprophyticus

11. GENUS: Staphylococcus
• Gram-positive
• (0.5 – 1 m)
• Non-motile, non-sporeforming
• Catalase (+)
• Facultative anaerobes

12. Medically important Staphylococci
• Staphylococcus aureus
• Staphylococcus epidermidis
• Staphylococcus saprophyticus

13. Staphylococcus aureus
• Catalase-positive, Coagulase-positive
• Salt tolerant (7.5% NaCl)
• Ferments mannitol on Mannitol salt agar

14. Reservoir
• Normal flora on nasal mucosa and skin

15. Transmission
• Spread via the hands and sneezing
• Fomites
• Surgical wounds
• Lungs of cystic fibrosis patients
• Foods associated with food poisoning
(Ham/Canned meats, Custard pastries and potato
salad)

16. Predisposing Factors for Infections
• Any break in skin (sx)
• Any foreign body (sx packing, sutures, tampons)
• Ventilators
• WBC <500/ L
• Dse: CF, CGD
• IV drug abuse

17. Virulence factors:
A. Cell associated factors
B. Extracellular factors

18. A) CELL ASSOCIATED FACTORS:
a) CELL ASSOCIATED POLYMERS
1. Cell wall polysaccharide (Peptidoglycan)
2. Teichoic acid
3. Capsular polysaccharide (some strains)
b) CELL SURFACE PROTEINS:
1. Protein A (S. aureus)
2. Clumping factor (bound coagulase)- S. aureus

19. Staphylococcal cell wall

20. B) EXTRACELLULAR FACTORS
a) Enzymes:
1. Catalase – all Staph.
2. Free coagulase (S. aureus)
3. Lipase
4. Hyaluronidase (Spreading factor)
5. β-lactamases
6. Staphylokinase (Fibrinolysin)
7. Proteinases

21. B) EXTRACELLULAR FACTORS
b) Toxins:
1. Cytolytic toxins
i) Hemolysins (α,β,γ,δ)
ii) Leucocidin (Panton-Valentine toxin)
2. Enterotoxin A-F (heat stable 60°C, 10 mins)
3. Toxic shock syndrome toxin-1 (TSST-1)
4. Exfoliative (epidermolytic toxin)
.

22. Staphylococcal Diseases
Infections
Intoxications

23. 1) Skin and soft tissue infections:
• Folliculitis, furuncle (boil), carbuncle, styes, abscess,
wound infections, impetigo, paronychia and less often
cellulitis.
• Coagulase

24. 2) Musculoskeletal
• Osteomyelitis, arthritis, bursitis, pyomyositis.

25. Osteomyelitis

26. 1. Respiratory: Tonsillitis, pharyngitis, sinusitis, otitis,
bronchopneumonia, lung abscess, empyema, rarely
pneumonia.
2. Central nervous system: Abscess, meningitis,
intracranial thrombophlebitis.
3. Endovascular: Bacteremia, septicemia, pyemia,
endocarditis.
4. Urinary: Urinary tract infection.

27. Infective endocarditis (Acute)
• Fever, malaise, leukocytosis, heart murmur (may be
absent initially)
• # 1 cause  S. aureus
• Fibrin platelet mesh, cytolytic toxins

28. B) INTOXICATIOINS:
The disease is caused by the bacterial exotoxins,
which are produced either in the infected host
or preformed in vitro.
There are 3 types-
1. Food poisoning
2. Toxic shock syndrome
3. Staphylococcal scalded skin syndrome

29. Gastroenteritis (food poisoning)
• 1-8 hours after ingesting toxin
• Nausea
• abdominal pain
• Vomiting
• followed by diarrhea
• No fever
• Enterotoxins A-F preformed in food (heat-stable)

30. Toxic Shock Syndrome (TSS)
• High fever (abrupt)
• Vomiting
• Diarrhea
• Myalgias
• Scarlatiniform rash
• Hypotension
• Cardiac/Renal failure
(severe cases)
• TSST-1

31. Staphylococcal Scalded Skin Syndrome
(SSSS)
• Exfoliative toxin

32. Laboratory Diagnosis:
Specimens collected: Depends on the type of infection.
• Suppurative lesion- Pus,
• Respiratory infection- Sputum,
• Bacteremia & septicemia- Blood,
• Food poisoning- Feces, vomit & the remains of
suspected food,
• For the detection of carriers- Nasal swab.

33. Treatment
• Methicillin/ Nafcillin/ Oxacillin/ Cloxacillin
• Methicillin-resistant S. aureus (MRSA) (due to changes
in major penicillin-binding proteins) is commonly
resistant to all antibiotics EXCEPT Vancomycin and
Fusidic acid.
• Topical mupirocin reduces nasal colonization.

34. Prevention
• Basic hospital infection control

35. Staphylococcus epidermidis
• Reservoir: skin and mucous membrane
• Neonatal Sepsis
• Peritonitis in patients with renal failure who are
undergoing peritoneal dialysis through an indwelling
catheter
• Most common  CSF shunt infection
• Infxn related to intravenous catheters and prosthetic
implants (e.g., heart valves, vascular grafts, and joints)
• Coagulase (-); Novobiocin (S)

36. Staphylococcus saprophyticus
• Causes U.T.I., particularly in sexually active young
women.
• 2nd cause community acquired U.T.I. young women
(Most common cause E. coli)
Coagulase (-); Novobiocin (R)

37. Characteristics S.aureus S.epidermidis S.saprophyticus
Coagulase + - -
Novobiocin Sensitive Sensitive Resistant
sensitivity
Acid from + - -
mannitol
fermentation
anaerobically
Hemolysis beta - (most) -

38. Laboratory Diagnosis
Gram stain: Gram-positive cocci
Differentiates
Staphylococc
us and Catalase test
Streptococcu
s
Staphylococcus Streptococcus
sp. sp.

39. Important Streptococci
• Streptococcus pyogenes
• Streptococcus agalactiae
• Enterococcus faecalis
• Streptococcus bovis
• Streptococcus pneumoniae
• Viridans group

40. Streptococcus
• Hemolysis varies by species:
•
•
•

41. Laboratory Diagnosis
  
OPTOCHIN BACITRACIN 6.5% NaCl
(S)S. pyogenes
(R)S. agalactiae
(S)S.pneumoniae (+)Enterococcus
(R)Viridans group ( - )S. bovis

42. Laboratory Diagnosis
• Optochin “Taxo P” Disc test

43. Laboratory Diagnosis
• Bacitracin “Taxo A” Disc test (S)

44. GENUS: Streptococcus
• Gram-positive
• Non-motile, non-sporeforming
• Catalase (-)
• Facultative anaerobes

45. Streptococci
• Are serogrouped using known antibodies to the cell wall
carbohydrates
(Lancefield’s Group A-H, K-U)
• Group A- Rhamnose-N-acetylglucosamine
• Group B-Rhamnose-glucosamine polysaccharide
• Group C-Rhamnose-N-acetylgalactosamine
• Group D- Glycerol teichoic acid
• Group F- Glucopyranosyl-N-acetylgalactosamine

46. Laboratory Diagnosis
• Specimen Collection & Processing:
• No special consideration; site
• Antigen Detection
• S. pyogenes  (throat) latex agglutination, Coagglutination,
ELISA
• Gram Stain

47. Laboratory Diagnosis
• Cultivation
• MOC: 5% Sheep’s Blood Agar (BAP)

48. Laboratory Diagnosis
• Colonial appearance
- Grayish white
- Hemolysis

49. Streptococcus pyogenes (GABS)
Distinguishing Characteristics
 Beta-hemolytic
 Group A
 Colonies inhibited by Bacitracin on BA
 Gram-positive cocci in chains
 Catalase-negative
 PYR (+)

50. Reservoir
• Human throat
• Skin

51. Transmission
• Spread by respiratory droplets
• Direct contact

52. Group A Streptococcal cell wall

53. Cell wall components
• Hyaluronic acid capsule (a polysaccharide) is non-
immunogenic; inhibits phagocytic uptake
• M-protein: major virulence factor, hair-like projections;
antiphagocytic, used to type group A Strep

54. Toxins
• Hemolysins
• Streptolysin O: immunogenic, hemolysin/cytolysin
• Streptolysin S: non-immunogenic, hemolysin/cytolysin

55. Exotoxins A-C
(pyrogenic/erythrogenic)
• Phage-coded (e.g., the cells are lysogenized by a
phage)
• Cause fever and the rash of Scarlet fever
• Inhibit liver clearance of endotoxin (from normal
flora), creating shock-like conditions
• Superantigens: activate many helper T cells by bridging
T cell receptors and MHC class II markers without
processed antigen

56. Spreading factors:
• Streptokinase (fibrinolysin): breaks down fibrin clot
• Streptococcal Dnase (Streptodornase): liquefies pus,
extension of lesion
• Hyaluronidase: hydrolyzes the ground substances of
the connective tissues; important to spread in cellulitis

57. Diseases
• Streptococcus pyogenes causes a wide variety of
acute infections; some have immunologic sequelae

58. Acute (Suppurative) S.pyogenes Infxn
• Pharyngitis (most common)
• Scarlet fever
• Pyoderma/ Impetigo
(Also, cellulitis,necrotizing fasciitis (flesh-eating bacteria!),
puerperal fever, lymphangitis, pneumonia, a toxic shock-
like syndrome, etc.

59. Pharyngitis
• Abrupt onset of sore throat
• Fever
• Malaise
• Headache
• Tonsillar abscesses
• Tender anterior cervical lymph nodes
• Lab: For Strep throat: Rapid antigen test (misses 25% of
the strep throat); culture all “negatives”

60. Pyoderma/ Impetigo
• Pus-producing skin infection (honey-crusted lesions)

61. Erysipelas
• Brawny edema, advancing margin of infection

62. Necrotizing fasciitis
• S. pyogenes  “flesh-eating bacteria”

63. Scarlet fever
• Initial: S/Sx’s of pharyngitis
• Followed by blanching, “sandpaper” rash

64. Scarlet fever
• Pastia lines

65. Scarlet fever
• Strawberry tongue

66. Non-suppurative Sequelae to Group
A Streptococcal Infections
• Rheumatic fever
• Acute glomerulonephritis (M12 serotype)

67. Rheumatic fever
• Sequelae to : Pharyngitis with group A Strep
(not group C)
• Mechanism: in genetically susceptible individuals, the
infection results in production of antibodies that cross-
react with cardiac antigens

68. Rheumatic fever
• Symptoms occurs 2-3 weeks after a pharyngeal
infection
• Lab: elevated ASO titers (>200)
• Jones Criteria

69. Major Jones Criteria
• “J NES”
• J- Joints (Migratory arthritis)
• -Carditis
• N- Subcutaneous Nodules
• E- Erythema marginatum
• S- Sydenham chorea

70. Minor Jones Criteria
• Fever
• Arthralgias
• Elevated acute phase reactants

71. Rheumatic fever
• DIAGNOSIS:
• 2 major or
• 1 major & 2 minor

72. Acute Glomerulonephritis
• Sequelae to: Pharyngitis or Cutaneous strep infxn
• Mechanism: Immune complexes bound to glomeruli

73. Laboratory Diagnosis
• PYR test
• Principle:hydrolysis of L-
pyrrolidonyl--
naphthylamide (PYR)

74. Treatment
• Penicillin G  DOC
• Beta-lactam drugs
• Erythromycin

75. Prevention
• Penicillin in RF px to prevent recurrent S. pyogenes
pharyngitis

76. Streptococcus agalactiae = Group B
Streptococci (GBS)
• Distinguishing Characteristics
• Beta-hemolytic
• Bacitracin-resistant on BAP
• Gram-positive cocci in chains
• Group B
• Catalase-negative, hydrolyzes hippurate
• CAMP test-positive: CAMP(Christie-Atkins-Munch-Peterson)
factor is a polypeptide that “compliments” a Staph aureus
sphingomyelinase to make an area of new complete beta-
hemolysis

77. Reservoir
• Colonizes human vagina (15 – 20% of women)

78. Transmission
• Newborn infected during birth
• Increased risk with PROM

79. Pathogenesis
• Beta-hemolysin

80. Diseases
• Neonatal septicemia
• Neonatal meningitis (Neonate – 2 mths)
• Most common causative agent ( GEL)
# 1 – S. agalactiae (GBS)
2 – E. coli
Rare: L. monocytogenes

81. Laboratory Diagnosis
• 0.04 U Bacitracin disk –
Resistant
• CAMP (Christie, Atkins,
Munch- Peterson) Test 
detects production of a
diffusible, extracellular
protein that enhaces
hemolysis of sheep
erythrocytes by S. aureus
• (+) Arrowhead shape at the
juncture of S. agalactiae & S.
aureus

82. Treatment
• Ampicillin with Cefotaxime or Gentamicin

83. Prevention
• Treat mother prior to delivery if she had a previous baby
with GBS, has documented GBS colonization, or
prolonged rupture of membranes

84. Streptococcus pneumoniae
(Pneumococcus)
• Distinguishing Characteristics
- Alpha-hemolytic
- Colonies inhibited by optochin on BAP
- Gram-positive, lancet-shaped diplococci
(or short chains)
- Lyse by bile
- Quellung (+)

85. Reservoir
• Human Upper Respiratory Tract

86. Transmission
• Respiratory droplets; not considered highly
communicable
• Often colonizes without causing disease

87. Pathogenesis
• IgA protease: colonization

88. Pathogenesis
• Teichoic acids: attachment
• Polysaccharide capsule: major virulence factor
• Pneumolysin O: hemolysin/cytolysin
• Damages respiratory epithelium (hemolysin similar to streptolysin
O, which damages eukaryotic cells)
• (Inhibits leukocyte respiratory burst and inhibits classical
complement fixation.)

89. Pathogenesis
• Pneumococcus in alveoli stimulate release of fluid and
red and white cells producing “rusty sputum”
• Peptidoglycan/ teichoic acids highly inflammatory in
CNS

90. Diseases
• Bacterial Pneumonia
• Adult Meningitis
• Otitis Media and Sinusitis in children
• Septicemia

91. Bacterial Pneumonia
• Most common bacterial cause, especially after 65 years
but also in infants
• Sx:
• “big” shaking chills
• Sharp pleural pain
• High fever
• Lobar with productive blood-tinged sputum (rusty-colored)

92. Predisposing Conditions for
Pneumonia
• Antecedent influenza or measles infection:
damage to mucociliary elevator
• Chronic obstructive pulmonary disorders
• Congestive heart failure
• Alcoholism
• Asplenia predisposes to septicemia

93. Adult Meningitis
• Most common cause (> 40 y/o)
• CSF: WBC (PMN)
Glucose Protein Pressure

94. Otitis Media and Sinusitis in Children
• Most common cause

95. Septicemia
• In splenectomized patients

96. Treatment
• Penicillin G  DOC
• Resistance (both low level and high level) is
chromosomal (altered penicillin-binding proteins); major
concern in meningitis (Vancomycin  Rifampin used)

97. Prevention
• Vaccine 23 serotypes of capsule

98. Viridans Streptococci (S. sanguis, S.
mutans, etc.)
• Distinguishing Characteristics
• Alpha-hemolytic, resistant to optochin
• Gram-positive cocci in chains
• NOT bile soluble

99. Reservoir
• Human oropharynx (normal flora)

100. Diseases
• Dental carries
• Infective Endocarditis (Subacute)

101. Dental carries
• S. mutans dextran-mediated adherence glues oral flora
onto teeth, forming plaque and causing caries

102. Infective Endocarditis
• Sx:
• Malaise
• Fatigue
• Anorexia
• Night sweats
• Weight loss
• Predisposing factors:
• Damage (or prosthetic) heart valve
• Dental work w/o prophylactic antibiotics
• Extremely poor oral hygiene

103. Pathogenesis
• Dextran (biofilm)-mediated adherence onto tooth
enamel or damaged heart valve and to each other
(vegetation). Growth in vegetation protects organism
from immune system.

104. Treatment
• Penicillin G with Aminoglycoside for endocarditis

105. Prevention
• For individuals with damage heart valve
• Prophylactic penicillin prior to dental work

106. GENUS: Enterococcus
• Catalase negative
• PYR +
• Hydrolyzes Esculin in
40% bile
• (+) growth 6.5% NaCl

107. Enterococcus faecalis =
Streptococcus faecalis
• Distinguishing Characteristics
• Group D Gram-positive cocci in chains
• PYR test +
• Catalase-negative, varied hemolysis
• Hydrolyzes esculin in 40% bile (bile esculin agar turns black)
• (+) growth 6.5% NaCl

108. Reservoir
• Human colon
• Urethra 
• Female genital tract

109. Pathogenesis/ Predisposing
Conditions
• Bile/ Salt tolerance allows survival in bowel and gall
bladder
• During medical procedures on GI or GU tract:
E. faecalis  bloodstream  previously damaged
valves  ENDOCARDITIS (SBE)

110. Diseases
• Urinary, biliary tract Infections
• Infective endocarditis (SBE)

111. Treatment
• All strains carry some drug resistance
• Some vancomycin-resistant strains of Enterococcus
faecium or E. faecalis: no reliably effective treatment

112. Prevention
• Prophylactic use of penicillin and gentamicin in patients
with damaged heart valves prior to intestinal or urinary
tract manipulation

113. Gram-negative cocci

114. GENUS: Neisseria
• Gram-negative
• Diplococci with flattened sides
• Oxidase positive

115. Important Genera
• Neisseria meningitidis
• Neisseria gonorrhoeae

116. Neisseria
Species N. meningitidis N. gonorrhoeae
Capsule
Pili
Vaccine
Portal of entry Respiratory Genital
Glucose Utilization
Maltose Fermentation
Oxidase test
Beta-lactamase prdxn Rare

117. Neisseria meningitidis
(meningococcus)
• Distinguishing Characteristics
• Gram-negative kidney bean-shaped diplococci
• Large capsule
• Grows on chocolate (not blood) agar in 5-10% CO2
• Ferments maltose
• Oxidase positive
• 13 Serogroups: A, B, C, D,29E, H, I, K,L,X,Y,Z & W-135

118. Reservoir
• Human nasopharyngeal area
• ≥ 5% carriers (asymptomatic)

119. Transmission
• Respiratory droplets
• Oropharyngeal colonization
• Spread to the meninges via the bloodstream
• Disease occurs in only small percent of colonized

120. Pathogenesis
• Important Virulence Factors
• Polysaccharide capsule (most impt)
• IgA protease allows oropharynx colonization
• Endotoxin (LPS): fever, septic shock in meningococcemia,
overproduction of outer membrane
• Pili and outer membrane proteins important in ability to
colonize and invade
• Deficiency in late complement components (C5-8) predisposes
to bacteremia

121. Diseases
• Meningitis
• Meningococcemia

122. Waterhouse-Friderichsen Syndrome
• Most severe form of meningococcemia
• High fever
• Shock
• DIC
• Ecchymoses
• Adrenal insufficiency
• Coma
• Death

123. Laboratory Diagnosis
• Specimen:
• Blood  culture only
• CSF  smear, culture (tube #2), chemical determination
• Petechial aspirate  smear/culture (?)

124. Laboratory Diagnosis
• G/S
• Presumptive dx: (+)
gram-negative cocci on
CSF smear
• Presumptive Dx: (+)
Oxidase test
(tetramethyl-para-
phenylenediamine-
dihydrochloride)

125. Laboratory Diagnosis
• Culture CAP 5-10% CO2
(candle jar)
 Incubate at 36-37 C at least 5
days before discarding as
negative
• Confirmatory test:
Carbohydrate Fermentation
test
• (+) Glucose
• (+) Maltose

126. Treatment
• Penicillin G – DOC
• Ceftriaxone
• β- lactamase production (rare)

127. Prevention
• Vaccine: capsular polysaccharide of strains
Y, W-135, C, A

128. Prophylaxis
• Rifampicin
• Ciprofloxacin

129. Neisseria gonorrhoeae
• Distinguishing Characteristics
• Gram-negative kidney bean-shaped
diplococci
• Intracellular Gram-negative diplococci
in PMNs from urethral smear is
suggestive of N.g.
• Sensitive to drying and cold

130. Reservoir
• Human genital tract

131. Transmission
• Sexual contact
• Birth

132. Pathogenesis
• Pili
• Attachment to mucosal surfaces
• Inhibit phagocytic uptake
• Antigenic (immunogenic) variation
• Most impt

133. Pathogenesis
• Outer membrane Proteins
• OMP I: Structural, antigen used in serotyping
• OPA proteins (opacity): antigenic variation, adherence
• IgA protease: aids in colonization and cellular uptake

134. Disease
• Gonorrhea

135. Laboratory Diagnosis
• Specimen
• Discharge from the GUT
• Discharge from the rectal mucosa
• Discharge from the throat/ oropharynx
• Skin lesions
• Eye/ Conjuntival Discharge
• Synovial Fluid

136. Laboratory Diagnosis
• Collection:
• Use Non-toxic cotton swabs (treated with charcoal to absorb toxic fatty
acid present in the cotton fiber)
• Swabs should be plated immediately (best method) or within 6 hours
• Specimen from sterile sites requires no special method in transport like
synovial fluids in the syringes, they should be transpotred immediately to
the laboratory
• Blood culture is an exception, N. gonorrheae and N. meningitidis are
sensitive to SPS (Sodium Polyanetholsulfate) which is present in
vacutainer tubes, if present should < 0.025%
• Transport media:
• Amie’s charcoal transport medium
• Transgrow medium
• New York City medium
• JEMBEC

137. Laboratory Diagnosis
• G/S & C/S of d/c
• Presumptive test – (+) gram-negative intracellular
diplococci
• Presumptive test – Oxidase test

138. Laboratory Diagnosis
• Culture
• Gold-standard in diagnosis
• Colonies: translucent, grayish, convex, shiny colonies with entire
margin, non-hemolytic
• TYPES:
• T1 & T2  Small, bright reflective colonies, typical of fresh
isolates from gonorrheae (+) fimbrae/pili
• T3, T4 & T5  Larger, flatter, non-reflecting
(-) fimbrae/pili

139. Media Used:
• Chocolate agar plate (CAP)
• Sterile sites
• Thayer-Martin Chocolate (T M) medium
• Modified medium of CAP
• Non-sterile sites
• Vancomycin, Colistin, Nystatin
• Modified Thayer-Martin (MTM)
• T-M + trimetroprim to (-) swarming Proteus
• M-Lewis Agar
• Same as T-M but instead of Nystatin, Anisomycin is use

140. Treatment
• Ceftriaxone – DOC
• Test for Chlamydia trachomatis or treat with tetracycline
• Penicillin-binding protein mutations led to gradual
increases in penicillin resistance from the 50s to the 70s
• Plasmid mediated β lactamase produces high level
penicillin resistance

141. Prevention
Adult forms: A B C
• Abstinence
• Be faithful/careful
• Condom

142. Prevention
• Neonatal:
• 0.5 % erythromycin ointment
• 1.0 % Tetracycline ointment
• 1.0 % Silver nitrate drops (Crede’s prophylaxis)

143. Moraxella catarrhalis
• Gram-negative diplococcus (close relative of neisseriae)
• Normal upper respiratory flora
• Otitis media
• Cause bronchitis and bronchopneumonia in elderly with
COPD
• Drug resistance a problem; most strains produce a β
lactamase