Staphylococcus

1. Staphylococcus
Mahsa jalili
MSc. Medical microbiology
1

2. 2

3. Historical perspective :
• Rosenbach (1884)
• Passat
• Zopf
• Evans and et al (1955)
• Silvestri and Hill (1965)
3

4. Historical perspective :
 Detection Staphylococcus of Micrococcus:
• Schleifer and Kandler cell wall
• Faller and et al cytochromes
• Nahaie and et al cellular fatty acid
• Kilpper and et al DNA-rRNAhybridization
• Ludwig and et al oligonucleotide16 srRNA
 Isolation of S.aureus bacillus subtilis :
• Gram stain
• low G + C content of DNA
4

5. DEFINITION OF THE GENUS :
• Shape : singly, pairs, tetrads ,short chains, grape-like clusters .
• nonmotile and nonspore-forming .
• More Species are anaerobic except S. aureus subsp. anaerobius and
Staphylococcus saccharolyticus (Obligatory anaerobic ).
• Growth in 10 percent sodium chloride and at 18-40 C .
• Staphylococci : Sensitive to furazolidone , resistant to Bacitracin
• ancient species Staphylococcus : S. succinus (tuberculostearic) :
coryneform bacteria.
5

6. CLASSIFICATION OF SPECIE
• The currently recognized species in the genus Staphylococcus
are shown in Table 32.1
6
S.aureus
S.auricularis
S.capitis
S.caprae
S.chromogene
S.Gallinarum
S.haemolyticus
S.hominis
S.hyicus
S.intermedius
S.Kloosii
S.Schleiferi
S.Sciuri
S.simulans

7. Recognition of species :
• According to :
 Using microcomplement fixation techniques
 Chemical properties of the cell wall
 ( Staphylococcus sciuri , Staphylococcus lentus,
Staphylococcusvitulinus ) : L-alanine residue
 Biochemical Properties : lactic acid , lactate
 Cellular fatty acid
 Physiological properties
 Colony morphology
 Molecular methods
7

8. Molecular methods
• ribotyping studies of Webster (1994)
• PFGE studies
• Polymerase chain reaction (PCR)
8

9. Sub specie :
phenotypic characters and DNA relatedness
9

10. Host range :
• host specilicity
• Compare species and sub-species
• ...
• S. xylosus
• S. caprae
10

11. Habitat
• Skin , sebaceous gland , mucous membranes.
• Divided into three groups in the skin .
• S. epidermidis (skin)
• S.epidermidis, S. hominis, and ,S. haemolyticus (children)
• S. capitis (S. capitis subsp. Ureolyticus )
• S. auricularis and S. capitis subsp. Capitis ( external auditory meatus)
• s.epidermidis and s.aureus (nose )
• S.epidermidis and S. hominis. S. schleifed (axillae and groins )
• S. lugdunensis (below the belt )
• S. capitis (groins )
11

12. Carriers
• People with
• diabetes mellitus insulin dependent
• Dialysis patients
• People who use intravenous drug.
• A blood group
12

13. Receptor
• Sites to connect :
• epithelial cells , Mucus , serum
• epithelial cells : Glycolipid , Glycoprotein , Proteoglycan
• Reducing connection :
• Increased secretion Lipoteichoic acid , ribitol teichoic acid
in streptococcal cell wall.
• Increased connectivity :
• mucus secretion
13

14. receptor for S. aureus
• In patients cystic fibrosis : asialoganglioside 1
(bronchial epithelial cell )
• In lung tissue : fucosylasialo - GM1, asialo-GMl, and asialo-
GM2
( glycolipids )
14

15. Points for adhesion
• 52152A/80/81 phage group complex :
colonization of newborn infants with staphylococc
• Increased connectivity in epithelial cells : PLs at
S. aureus and AAP at S. epidermidis .
• Increased connectivity in epithelial cells : Clf B in
S. aureus .
15

16. Point
S.
epidermidls
Corynebacterium S. aureus
16

17. Light microscopy and staining reacilons
• young cultures : (18-24 h) - diameter of 0.5-1.5 pm
• old cultures : ( >48 h ) -
• The Colony :
 S. aureus
 S. hominis, S. haemolyticus, and S. capitis
 S. saprophyticus and S. cohnii
• Cell wall defective :
 S.aureus and S. epidermldls (L-form)
17

18. L-form
18
The body fluids of patients with
immunodeficiency
Osmotic pressure-sensitive
Non-Coloring with Gram stain
No growth in normal culture medium
Chronic infections and malignant
tumors

19. Colony morphology
• media : variety of commercial :
• selective
• nonselective
19
Selective media
Mannitol salt
agar
lipase-salt-
mannitol agar
Phenyl ethyl
alcohol
agar
Baird-Parker agar (CNA)

20. media for the primary isolation
• Blood agar with sheep blood (35-37'C ) , (48-72 h )
• tryptic soy agar
• BHI
• tryptose phosphate broth
• Mannitol salt agar
• Mannitol salt Lipase
• Phenyl ethyl alcohol agar
• Columbia colistin-nalidixic acid (CNA) agar
• Brewer's thioglycollate medium within (24-12 h) at (35-37'C )
20

21. media
• Baird- parker agar :
 For identify S. aureus in food
 Prevent the growth of Gram-negative bacteria
• Tryptic soy agar and P agar :
 For cultivation of various species of Staphylococcus aureus from skin
• Schleifer- Kramer agar :
 S. aureus isolated from food and pollution sources
 Incubation (48-72 h) at (35-37'C )
21

22. Exception
• Different species of staphylococci :
• Size . turbidity . Pigment . borders of colony .
• Staphylococcus epidermidis :
• Two or more morphology of the colonies
22

23. • one explanation for this phenomenon :
• ica operon with encoding the production of polysaccharide intercellular
adhesin (PIA)
• mediates intercellular adherence of bacteria
• the accumulation of a multilayered biofilm.
23

24. pigment
• Conditions pigment production :
• The best temperature for pigment production : 22 °
• Solid media and under aerobic conditions
• Color pigments : Yellow
• Blood agar with sheep blood : pink to yellow range
24

25. pigment
25
Deviolet , pinkish , or brownish
pigment.
S. epidermidis, S. intermedius, and S.lentus
S. vitulus, S. lentus, S. caseolyticus, and some
strains of S. sciuri,
grow poorly on P agar.

26. The main pigment in staphylococci : staphyloxanthin
26

27. Growth in broth and semi solid media
• typtic soy broth
• brain-heart infusion broth
• nutrient broth
• tryptose phosphate broth
• Species such as S. aureus,S. epidermidis, S. lugdunensis, and S. schleiferi,
produce abundant anaerobic growth with over night incubation.
• S.hominis, S. auricularis, S. kloosii, S. equorum, S. arlettae,S. vitulinus, and
S. lentus fail to grow, or slowly, in the anaerobic portion of this medium.
27

28. Important note
• abnormal morphology :
• 1. Capsule in some strains.
• 2. L-form
• 3. SCV ( small colony variants )
28

29. Character of SCV
 Resistant to aminoglycosides
 Slow growth
 absence of pigmentation
 reduced range of carbohydrate utilization,
 failure to virulence factors
 Two classes of SCVs :
• 1. stable following serial passage
• 2. revert back to wild type
 Switching between gentamicin resistant SCV and gentamicin
sensitive wildtype : genetic mutation.
 In human infection : prove resistant to antibiotic cure
 mutation in genes involved in the electron transport chain :
that become auxotrophs
29

30. METABOLISM
30

31. Carbohydrate metabolism
• Lactic acid and lactate are major sources of carbon .
• These substances can enter the path pyruvic acid .
 Staphylococci are capable of using a variety of carbohydrates
as carbon and energy sources .
31

32. carbohydrate
without a change in
its form
modified during
uptake
32

33. phosphotransferase system
• Glucose,
• mannose,
• glucosamine,
• fructose,
• lactose,
• galactose,
• mannitol,
• N-acetylglucosamine,
• and B-glucosides
33

34. lactose-specific PTS
• composed of four enzymes :
 enzyme I (El)
 Hpr
 enzyme lll (EIll)
 enzyme Il (EII)
• Some carbohydrates are not taken up via the PTS :
 pentoses ribose,
 xylose,
 and arabinose,
 and the corresponding pentitols
34

35. glucose metabolism
• two central routes used by staphylococci :
 The Embden-Meyerhof-Parnas (EMP) (glycolytic)
 S. aureus and S. epidermidis
 oxidative hexose monophosphate pathway (HMP)
35

36. glucose metabolism
• S. aureus
• The major product of anaerobic glucose metabolism :
 lactate
 acetate
 pyruvate
• The major product of aerobic glucose metabolism :
 acetate
 CO2
36

37. glucose metabolism
• S. epidermidis
• The major product of anaerobic glucose metabolism:
 Lactate
• The major product of aerobic glucose metabolism :
 acetate
 CO2
 formate
• S. saccharolyticus
• The major product of anaerobic glucose metabolism:
 Ethanol
 formic acid
 and CO2
• The major product of aerobic glucose metabolism :
 lactic and
 formic acid
37

38. CELLULAR
COMPONENT
38

39. Cell membrane
• phospholipids and proteins.
•
• Functions : electron transport .active transport, ....
• components of the electron transport system : cytochromes and
menaquinones
• types of cytochromes :a.b.c
• menaquinones :MK6 ,…,MK9
39

40. Point
• Isoprenoides MK6
 S. hyicus
 S. equorum
 S. sciuri
• Isoprenoides MK8
 S. aureus
 S. lugdunensis
40

41. Cell membrane
• penicillin-binding proteins (PBP)
• The main pigment is staphyloxanthin
• Two siderophores are known to be produced :
• staphyloferrin A ,
• staphyloferrin B
41

42. Cell wall
• Peptidoglycan and teichoic acid.
• N-acetylglucosamine and N-acetylmuramic acid.
• D-amino acids in staphylococci : L-lysine
(S.succinus)
• Interpeptide bridges
42

43. Cell surface associated and secreted adhesins
• In S. aureus cell wall-associated protelns …
• play role in colonization and invasive disease .
• A total of 21 genes encoding surface proteins :
 protein A SpA
 Collagen - binding protein (Cna )
 the fibrinogen - binding proteins clumping factor A and B
(ClfA and ClfB)
 two flbronectin-binding proteins FnBPA and FnBpB .
43

44. Cell surface associated and secreted adhesins
• Other less well characterized proteins include :
 serine-aspartate multigene family (SdrC, SdrD, and SdrE)
 the bone sialoprotein-binding protein Bbp
 The plasmin-sensitive protein Pls .
 biofilm-associated protein (Bap) .
Protein expression is influenced by at least three
major regulatory systems : Agr . Sar . Sae
44

45. Cell surface associated and secreted adhesins
• Fibronectin
• Collagen - binding protein (Cna )
• serine-aspartate multigene family
• bone sialoprotein-binding protein
• plasmin-sensitive protein
• biofilm-associated protein
45

46. 46

47. Exocellular material
• Staphylococci produce a variety of different extracellular .
• S. aureus,
• Toxins
 cytolytic toxins : hemolysin (alpha-, beta-, delta-, gamma-)
 Superantigen : (enterotoxin, exfoliative toxin , TSST-1 )
47

48. 48

49. 49

50. Gene regulation
• Environmental conditions
• Exponential phase
• Post exponential
• Stationary phase
• Gene regulator : agr , sar , sae
• Agr : Including two promoter (P2 , P3 )
• P2 : agr BCDA
• P3 : RNA III
• S. aureus = S. epidermldls ,S. warnery , s. simulans
• Sar gene : Secretory protein
50

51. Bacteriocins
• Lantibiotics
 Staphylococci , Iactococci, bacilli , and streptomycetes
• Small polypeptides ) 19-34 amino acids (
• sulfide rings ) meso-lanthionine and 3-methyllanthionine (
 Lantibiotics produced by S. epidermldls : epidermin , Pep5
 S. gallinarum : Gallidermin
51

52. Plasmids
• Plasmids are facultative extrachromosomal genetic
systems (elements) .
• S.aureus plasmids classified into three classes .
52

53. Class I plasmids
• small size (1-5 kbp) .
• high copy number .
• encode a single antibiotic resistance.
• Widespread in the genus Staphylococcus.
• divided into four families :
 TP 181 , PC 194 ,PSN 2 , PC 194
• resistance to tetracycline , Chloramphenicol , Macrolides
• Without transposon
• Reproduction : Rolling circle
53

54. Class II plasmids
• Divided into two major families on the basis of
incompatibility.
• are relatively large (15-33 kbp) .
• Low copy number .
• using the θ replication mechanism .
• Genes penicillinase and lactamase .
• Resistance genes in metals.
• Transposon
• Uncommon
54

55. Class III plasmids
• dentified in many other staphylococcal species.
• unlike class I
• Very uncommon in the species S. hyicus, S. intermedius, S.
simulans, and S. lugdunensis
• conjugative plasmids
• Divided into three groups : A,B, C
 B : Transformation
55

56. Resistance to antibiotics
• Penicillin
• Methicillin : MRSA , MRSE
• Three categories of S. aureus resistance to vancomycin
described in 1997 :
 1. (VISA)
 2. (hvisA )
 3. (VRSA)
• developed by the (CDC) for the identification of VISA. These
are :
 growth BHI and containing 6 ugml vancomycln
 E-test
 broth microdilution
56

57. Resistance to antibiotics
• Erythromycin
• Cross resistance to ( MLS )
• Macrolide ,lincosamide, and streptogramin
• Tetracycline
 1.tet k
 2.tet M
• Aminoglycoside
• 1. Mutations
• 2. Permeability
• 3. modifying
57

58. Resistance to antibiotics
• trimethoprim
 dfrB of S. aureus
 dfrC of S. epidermidis
• fluoroquinolone
 S. aureus and S. epidermldls (gyrA )
 S. aureus, S.epidermidis, and S. haemolyticus (norA)
 S. aureus (grlA )
58

59. Resistance to antibiotics
Plasmid
• Penicillin
• Tetracycline
• Aminoglycoside
• Erythromycin
Chromosome
• Methicillin
• Vancomycin (VISA)
• Vancomycin (VRSA)
59

60. Resistanceto other compound
 S. aureus,
 S. lugdunensis
 S. epidermidis
 S. saprophyticus
 Cadmium
 zinc
 bismuth
 Lead
 Mercury
 arsenate
 arsenite
 antimony
60

61. isolation of staphylococci
61

62. isolation of staphylococci
• DNA ase Test
• Oxacillin agar
• Latex agglutination test
• Screen test – MRSA
• PCR
62

63. Typing methods…
in the past
• Phage typing
Nowadays
• Ribotyping
• PFGE
• multilocus enzyme
electrophoresis (MLEE)
• Plasmid profiling
• multilocus sequencetyping
(MLST)
63

64. S.aureus diseas
• Determinants of infection can be divided into those
associated with host and bacterial factors .
• rols of host
• genetic susceptibility
• People with immune deficiency
• Receptors on endothelial cells
• Artificial means in the body
• role of bacteria
• Virulence factors
• process of invasion
64

65. S.aureus diseas
• wide range of infections.
• Community-acquired infections
 toxin-mediated diseas
 infections the skin and soft tissues
 infection of bones and joints
 infection relating to other deep sites
 abscess formation in liver, spleen, ….
• Nosocomial or hospital-acquired infections .
 wound infection
 ventilator-associated pneumonia
 bacteremia
65

66. Bacteremia and endocarditi
• After local infection.
 Nosocomial infection
 Community-acquired infections
 other things : People with diabetes, Cardiovascular disease ,
Immunodeficiency
 Symptoms :Fever . Chills .
 Complications : endocarditis , Metastatic infection
66

67. Pneumonia
• After influenza
• in cases :
 The epidemic influenza
 In newborns with cystic fibrosis and with measles
 Immunodeficiency
 High mortality
• Pneumonia
 Primary pneumonia
 Secondary pneumonia
67

68. Toxin-mediated staphylococcal disease
• Food poisoning
 S. aureus is inoculated from a skin lesion into food .
 The reaction began with vomiting
 antibiotic treatment is not necessary.
• Staphylococcal scalded skin syndrome (SSSS )
 exfoliative toxins in S.aureus.
 Neonates and young children.
 Nikolsky's sign
68

69. Cutaneous infection
• The most common bacterial skin infection.
• Paronychia
• Folliculitis (sycosis barbae , sycosis vulgaris )
• Furuncle
• Impetigo
• (ssss)
 Ritter s disease
 Bullous impetigo
 Staphylococcal scarlet- fever
69

70. Vaccination
• S. aureus
type 5 and 8 capsular polysaccharides conjugated to
nontoxic recombinant Pseudomonas aeruginosa exotoxin .
70