Gram positive bacteria

1. Staphylococcus
spp

2. Introduction
• Staphylococcus and Micrococcus are gram
positive cocci
• Other gram positive cocci includes Streptococci,
Enterococcus
• Presence or absence of catalase is used to divide
gram positive cocci into various genera
• Staphylococci and micrococcus are catalase
positive.

4. cont
• Staphylococci are similar to Micrococci in shape
• But Staphylococci can be:
a) pathogenic
b) commensal
c) Oxidative & fermentative

5. While Micrococci are:
a) Commensal (Normal flora of skin)
b) Only oxidative (Non fermentative)
• Can be differentiated from Staphylococci by oxidation
fermentation reactions (O – F) test

6. Staphylococcus
 Staphylococci are gram positive cocci,
facultative anaerobes, catalase positive
 Occur in grape like clusters
 In Greek Staphylé =“bunch of grapes”
(kokkus= grain or berry)

8. CLASSIFICATION:
A) Based on coagulase production:
1. Coagulase positive: Eg- S. aureus
2. Coagulase negative: Eg- S. epidermidis
S. saprophyticus
B) Based on pathogenicity:
1. Common pathogen: Eg- S. aureus
2. Opportunistic pathogens: Eg- S. epidermidis, S.
saprophyticus
3. Non pathogen: Eg- S. homonis

9. Staphylococcus aureus
MORPHOLOGY:
• These are spherical cocci.
• Approximately 1μm in diameter.
• Arranged characteristically in grape like
clusters.
• They are non motile and non sporing.
• A few strains possess capsules.

10. Culture
Media used :-
i) Non selective media: Nutrient agar,
Blood agar,
MacConkey’s agar.
ii) Selective and differential media: mannitol salt agar
• Facultative anaerobes and able to grow on media
containing high concentration of salt (10% NaCl)
• Grow best at 35 – 37 ⁰C, but can grow 10-47⁰C.

11. Cultural Characteristics:
i) On nutrient agar- The colonies are large,
circular 2-3 mm in diameter ,convex, smooth,
shiny, opaque
• Most strains produce golden yellow
pigments after prolonged incubation

12. ii) On MacConkey’s agar- The colonies are small & pink in
colour.
iii) On blood agar- Most strains produce β- haemolytic
colonies.

13. Biochemical reactions:
• 1) Catalase test- Positive.

14. 2) Coagulase test-
i) Slide coagulase test- Positive.
ii) Tube coagulase test- Positive.
Slide coagulase test Tube coagulase test

15. Biochemical test
• 3) DNAse thermostable nuclease that break down DNA
• 4) Ferments mannitol anaerobically with acid only

16. Pathogenesis
Source of infection:
A) Exogenous: patients or carriers
B) Endogenous: From colonized site
Mode of transmission:
A) Contact: direct or indirect( through fomites)
B) Inhalation of air borne droplets

17. Pathogenesis
Pathology depends on:
• Production of surface proteins that mediate
adherence of bacteria to host tissue
• Elaboration of extracellular proteins such as
specific toxins and hydrolytic enzymes

18. Virulence factors:
These include
A) Cell associated factors
B) Extracellular factors

19. A) CELL ASSOCIATED FACTORS:
a) Cell associated polymers
b) Cell surface proteins
a) CELL ASSOCIATED POLYMERS
1. Capsular polysaccharide
2. Peptidoglycan
3. Teichoic acid
b) CELL SURFACE PROTEINS:
1. Protein A
2. Clumping factor (bound coagulase)

21. B) EXTRACELLULAR FACTORS
a) Enzymes
b) Toxins

22. a) Enzymes:
1. Free coagulase
2. Catalase
3. Lipase
4. Hyaluronidase
5. DNAase
6. Staphylokinase (Fibrinolysin)
7. Phosphatase

23. b) Toxins:
1. Cytolytic toxins (membrane
damaging toxins
Alpha haemolysin
Beta haemolysin
Gamma haemolysin
Delta haemolysin
Leucocidin (Panton-Valentine toxin)
2. Enterotoxin (8 serologically toxin)
3. Toxic shock syndrome toxin (TSST-1)
4. . Exfoliative (epidermolytic toxin)

24. Clinical diseases:
Diseases produced by Staphylococcus aureus
occurs through
A) proliferation of the organisms ,leading to abscess
formation and tissue destruction (infections)
B) Intoxications (toxin production)

25. A) INFECTIONS:
Mechanism of pathogenesis:
Cocci gain access to damaged skin, mucosal or
tissue site
Colonize by adhering to cells or extracellular matrix
Envade the host defense mechanisms and multiply
Cause tissue damage

26. Common Staphylococcal infections are:
1) Skin and soft tissue:
• Impetigo - localized cutaneous infection characterized
by pus filled vesicle
• Folliculitis- pyogenic infection in the hair follicles (
styes=eyelid)
• Furuncles (boil)- large, painful, pus-filled cutaneous
nodule
• Carbuncles – coalescence of furuncles with extension
to subcutaneous tissue

27. Impetigo
Folliculitis
Folliculitis

28. Styes Abscess

29. Furuncle (boil)
Carbuncle

30. • 2) Musculoskeletal:
• Osteomyelitis: destruction of the long bones, particularly
the metaphyseal area
• Septic arthritis: Painful erythermatous joint with collection
of purulent material

31. 3.Respiratory infections:
• Pneumonia: primarily in the very young, elderly and
patients with underlying lung diseases
• Empyema: occur in 10% of patients with S.aureus
pneumonia
4. Cardiovascular : Bacteremia, Endocarditis

32. B) INTOXICATIOINS (Toxin production):
• The disease is caused by the bacterial exotoxins,
which are produced either in the infected host
or preformed in vitro rather than direct effect of organisms.
There are 3 types-
• Food poisoning
• Toxic shock syndrome
• Staphylococcal scalded skin syndrome

33. 1) Food poisoning:
 Enterotoxin is responsible for manifestations of
staphylococcal food poisoning.
 Eight types of enterotoxin are currently known,
named A, B, C1-3, D, E, and H.
 It usually occurs when preformed toxin is ingested
with contaminated food.

34.  The common food items responsible are - milk and milk
products, meat, fish and ice cream.
 Source of infection- food handler who is a carrier.
 Incubation period- 2 to 6 hours.
 Clinical symptoms- nausea, vomiting and diarrhoea.
 The illness is usually self limited, with recovery in a day or so.

35. 2) Staphylococcal Toxic shock syndrome (STSS):
• STSS is associated with infection of mucosal or
sequestered sites by TSST- 1 producing S.aureus.
• It is fatal multi organ systems disease presenting with
fever, hypotension, vomiting, diarrhoea, mucosal
hyperemia and erythematous rash which desquamates
subsequently.

36.  2 types of STSS known (staphylococcal toxic shock
syndrome)
i) Menstrual associated STSS: Here colonization of
S.aureus occurs in the vagina of menstruating woman
who uses highly absorbent vaginal tampons.
ii) Non menstrual associated STSS: Here colonization of
S.aureus occurs in other sites like surgical wound.

38. 3) Staphylococcal scalded skin syndrome (SSSS):
• Exfoliative toxin produced by S.aureus is responsible for
this
• It is a skin disease in which outer layer of epidermis
gets separated from the underlying tissues
• Development of a painful rash, slough off; the skin
resembles scalding

40. Lab diagnosis
• Specimen collected depends on the type of
infections discharge eg pus.
- Suppurative lesion- Pus
- Respiratory infection- Sputum
- Bacteremia & septicemia- Blood
- Food poisoning- the remains of suspected
food
- For the detection of carriers- Nasal swab

41. 1) Direct microscopy:
• Direct microscopy with
Gram stained smear,
cocci in clusters are
seen
• This is of no value for
specimen like sputum
where mixed flora are
normally present

42. c) Gram staining: Smears are
examined from the culture
plate and reveals Gram
positive cocci(1μm in
diameter) arranged in
grape like clusters.
II) Culture:
a) Media used:
b) Cultural Characteristics:

43. d) Biochemical reactions
4) Antibiotic sensitivity tests done as a guide to treatment
5) Bacteriophage typing is done for epidemiological
purposes
6) Serological tests are not useful

44. Treatment
 Drug resistance is common
 Penicillin used to be treatment of choice, but 90%
of hospital strains are resistance
 Resistance is due to production of Beta-
lactamase enzymes.
 Semisynthetic penicillins ( methicillin, cloxacillin) is
used against beta-lactamase producing strains
 Methicillin Resistant Staphylococcus aureus
(MRSA) strains have become common

45. Treatment
 Currently, vancomycin is the treatment of
choice for MRSA
 Unfortunately, isolates have now developed
resistance to vancomycin.

46. Epidemiology
• Normal flora on human skin and mucosa surfaces
• Acquisition may be exogenous or endogenous
• Hospital infections by staphylococci deserve
special attention because of their frequency &
they are caused by strains resistant to various
antibiotics
• Common cause of postoperative wound infection
and other hospital cross infections

47. Prevention
 Spread of staphylococci from person to
person is more difficult to control
 Proper hand washing and the covering of
exposed skin surfaces
 MRSA also difficult to control because
asymptomatic nasopharyngeal carriers are
common source of infections.

48. Coagulase –Negative
staphylococci
• Catalase positive, coagulase negative, gram
positive cocci arranged in clusters
• Facultative anaerobes
• Are opportunistic pathogens that causing
infection in debilitated or compromised
patients
• Often colonizing biomedical devices
• Includes: S.epermidis, S.saprophyticus

49. Staphylococcus
epidermidis
• Major pathogen in involving prosthetic
implants such as intravascular lines or
cardiac valves
• Pathogenesis involves biofilm
production
• Endocarditis may be caused,
particularly in drug addicts

50. Staphylococcus
saprophyticus
 It causes urinary tract infections, mostly in
sexually active young women
 Men are infected much less often
 It is one of the few frequently isolated that is
resistant to Novobiocin

51. Distinguishing tests between
S.aureus, S.epidermidis and
S.saprophyticus
Characters S.aureus S.epidermidis S.saprophytic
us
Coagulase + _ _
DNAse + _ _
Mannitol
fermentation
+ _ _
Phosphotase + + _
Novobiocin + + _

52. Thank you