This document provides information about Staphylococcus including its taxonomy, history, virulence factors, pathogenesis, and clinical manifestations. Staphylococcus is a genus of bacteria that can be found commonly on the skin and nose of humans. Staphylococcus aureus is an important human pathogen able to cause a variety of infections through factors such as its capsule, toxins, and ability to evade the immune system. Common infections include skin and soft tissue infections, bone and joint infections, and pneumonia. Laboratory diagnosis involves culturing samples on nutrient agar to identify characteristic golden yellow colonies of staphylococci.

1. Staphylococcus
Dr. Abhijeet Mane
Assistant Professor
BVDUMC
Pune
Thursday, October 13, 2016 1

2. Index
 Taxonomy
 Introduction
 History
 Staphylococcus aureus
 Factors predisposing serious infection
 Virulence factors
 Pathogenesis
 Clinical Manifestations
 Laboratory Diagnosis
 Detection of MRSA
 MRSA Control measures
Thursday, October 13, 2016 2

3. Taxonomy
 Staphylococci are Gram positive cocci
and classified into 2 families (as per
1986 edn. Of Bergey’s Manual of
Systemic Bacteriology):
◦ Micrococcaceae (GPC, catalase positive)
◦ Streptococcaceae (GPC, catalase negative)
 Micrococcaceae included 4 genera
◦ Planococcus
◦ Micrococcus
◦ Stomatococcus
◦ Staphylococcus
Thursday, October 13, 2016 3

4.  Newer edition suggests: Staphylococci
are
 Phylum: Firmicutes
 Genus I
 Family V : Staphylococcaceae
 Order I : Bacillales
 Class III : Bacilli
Thursday, October 13, 2016 4

5. Introduction
 Staphylococci are non motile, non
spore forming, catalase positive, GPC
 Arranged in single cells, pairs, tetrads,
short chains but appear predominantly
in grape like clusters
 Facultative anaerobes, except
S.aureus subsp. anaerobius and S.
saccharolyticus
(These are often CATALASE –
NEGATIVE)Thursday, October 13, 2016 5

6.  Staph: generally found on skin, mucous
membrane of humans and other animals
 Other colonisation sites: intertriginous skin folds,
perenium, axillae, vagina
 Sometimes this association is amazingly specific:
◦ S.capitis ssp capitis: normal human flora of skin and
seb. Glands of scalp, forehead, neck
◦ S.auricularis: external auditory canal
◦ S.hyicus: infectious dermatitis in swine
 Some pathogenic Staphylococci produce –
Coagulase – detected in lab
 S.aureus and CONS (S.epidermidis,
S.saprophyticus) seen frequently in human
infections
Thursday, October 13, 2016 6

7. History
 1st observed in pus: von Recklinghausen
(1871)
 1st cultured in liquid media: Louis Pasteur
(1880)
 Named ‘Staphylococcus’ (Gr. Staphyle –
bunch of grapes, kokkos - berry) by Sir
Alexanger Ogston (1880)
 Rosenbach (1884) – named S.aureus
(golden yellow colonies), S.albus (white
colonies)
 Passet (1885) – S.citreus (lemon yellow
colonies)
Thursday, October 13, 2016 7

8. Thursday, October 13, 2016 8

9. Staphyloccus aureus
 Catalase – positive, coagulase –
positive, facultative anaerobe, non
motile, non sporing, occasionally
capsulated
 Spherical cocci, 1 micron diameter,
arranged in grape like cluster (due
to…)
 Produces golden yellow pigment on
NA and beta hemolytic colonies on
SBA
Thursday, October 13, 2016 9

10. Thursday, October 13, 2016 10

11. Factors predisposing serious
infections
 Defects in leukocyte chemotaxis, either
congenital (Wiskott AS, Down’s, Job’s
syndrome) or acquired (DM, RA)
 Defects in opsonisation by antibodies
secondary to congenital or acquired
hypogammaglobulinemia
 Defects in complement component (esp.
C3 & C5)
 Defects in intracellular killing of bacteria
following phagocytosis
Thursday, October 13, 2016 11

12.  Skin injuries (eg. Burns, surgical
incisions, eczema)
 Foreign bodies (sutures, i/v lines,
prostheses)
 Infection with other agents (eg.
influenza)
 Chronic underlying diseases
(malignancy, alcoholism, heart
disease)
 Therapeutic or prophylactic AMAThursday, October 13, 2016 12

13. Burns
Suture site infection
iv line
infection
Thursday, October 13, 2016 13

14. Virulence factors
 Capsular polysaccharide
 Peptidoglycan and Teichoic acids
 Protein A
 Enzymes
 Hemolysin
 Toxins
 Superantigens
Thursday, October 13, 2016 14

15.  Capsular polysaccharide
 Production of an exopolysaccharide
 Prevents ingestion of organism by PMN
cells
 Seen by EM studies of – infected
pacemaker leads, peritoneal catheters,
i/v lines
 Clinical isolates of SA – classified into 11
types based on capsular polysaccharide
immunotyping
 From significant clinical isolates:
capsular serotype 5 or 8 isolated
 These 2 types also associated withThursday, October 13, 2016 15

16.  Peptidoglycan and teichoic acid
 SA cell wall contain peptidoglycans
 Crossed linked polymers of N-acetyl-
glucosamine and N-acetyl-muramic
acid
Thursday, October 13, 2016 16

17.  Teichoic acids function:
◦ specific adherence of SA to mucosal surfaces
◦ Provide rigidity and resilience to cell wall
◦ Activate complement, enhance chemotaxis of
PMN cells, elicit production of IL-1 by
monocytes, stimulate production of opsonic
Abs
 Several other proteins including
adhesins, fibronectin binding proteins,
collagen binding proteins, clumping
factor covalently incorporated in
structure of SA peptidoglycan
Thursday, October 13, 2016 17

18.  Protein A
 MW – 42 kDa
 Found on cell surface and in growth medium
 Able to bind with Fc region of all human IgG subclasses
except IgG3
 Interferes with opsonisation, ingestion of organism by
PMN cells, activates complement, elicits immediate and
delayed hypersensitivity reactions
 It is immunogenic; Abs are found in pt with serious SA
infections
 Presence of protein A provides basis for Co-
agglutination reaction (used to identify gonococci,
streptococcal grouping) and detection of bacterial Ags
in body fluids
Thursday, October 13, 2016 18

19.  Enzymes
◦ Catalase:
 Function to inactivate toxic H2O2 and free
radicals formed by myeloperoxidase system
within phagocytic cells after ingestion of SA
◦ Clumping factor:
 Cell bound material able to bind fibrinogen,
responsible for binding of SA to both fibrin &
fibrinogen
Thursday, October 13, 2016 19

20.  Coagulase:
◦ Can exist in free or bound form
◦ Binds to prothrombin – becomes active
◦ Catalyses conversion of fibrinogen to
fibrin
◦ Leading to coat bacterial cell with fibrin,
rendering them more resistant to
opsonisation and phagocytosis
 Fibrinolysin
◦ Breaks down fibrin clots and facilitate
spread of infection to contiguous tissues
Thursday, October 13, 2016 20

21.  Hyaluronidase
◦ Hydrolyses intercellular matrix of acid
mucopolysaccharides in tissues – spread
organisms to adjacent areas in tissues
 Lipases
◦ Seen in chronic furunculosis pt
◦ Spread organism in cutaneous and sub
cut tissues
Thursday, October 13, 2016 21

22.  Phosphatidylinositol – specific
Phospholipase C
◦ Associated with ARDS and DIC pt
◦ Tissues become more susceptible to
damage & destruction by bioactive
complement components and products
during complement activation
 Nuclease and phosphodiesterase
◦ Having exonuclease and endonuclease
activity
Thursday, October 13, 2016 22

23.  Beta-lactamase enzymes:
◦ Inducible (produced only in presence of
beta lactam AMA) or
◦ Constitutive (produced continually)
 Resistant to penicillin and ampicillin
 Plasmid coded
 Resistance to several AMA like erythromycin
and tetracycline
 Resistance may be transferred by
Transformation and Transduction
Thursday, October 13, 2016 23

24.  Hemolysin : Alpha hemolysin
 Lethal effects on human PMNs cells
 Lyse rabbit RBCs
 MW – 33 kDa
 Secreted in medium : late logarithmic growth phase
 Mech. Of Action:
◦ Individual monomers interact on target cell membrane to form
cylindrical heptamers with central pore
◦ Pores allow rapid efflux of potassium ions and influx of Sodium
and Calcium ions
◦ Leads to osmotic swelling and rupture of cell
 Also dermonecrotic on s/c inj
 Neurotoxicity: demyelination of both rabbit and murine
models
 Zone of hemolysis on SBA
Thursday, October 13, 2016 24

25.  Beta hemolysin
 A sphingomyelinase
 Exotoxin, MW – 35 kDa
 Secreted toward end of logarithmic
growth phase
 Exhibits “hot-cold phenomenon”:
◦ Maybe due to initial disruption of cohesive
forces within membrane by toxin and
subsequent phase separation within
membrane itself as temp is lowered
 Synergy – with CAMP factor; identify Gr.
B Streptococci
Thursday, October 13, 2016 25

26. Thursday, October 13, 2016 26

27.  Delta hemolysin
 MW – 3 kDa
 Secreted toward end of exponential
growth phase
 Produced by > 97% SA strains
 MOA:
◦ Acts as surfactant;
◦ disrupts cell membrane
◦ Forms channels;
◦ leakage of cellular contents
Thursday, October 13, 2016 27

28.  Gamma hemolysin:
 Has 3 protein fragments which act
together with Leukocidin to exhibit
hemolytic activity
 MOA:
◦ Exerted on PMNs membranes causing
degranulation of cytoplasm
◦ Cell swelling leading to cell lysis
◦ Formation of pores seen
Thursday, October 13, 2016 28

29.  Toxins:
 Exfoliatin or epidermolytic toxin
 Made up of 2 proteins
 ET-A and ET-B
◦ ET-A – thermostable, structural gene
chromosomal
◦ ET-B – heat labile, plasmid origin
 Proteolytic, dissolve mucopolysaccharide
matrix of epidermis
 Results in intra epidermal splitting of cellular
linkages in the stratum granulosum
 SSSS
Thursday, October 13, 2016 29

30.  Enterotoxin:
 A, B, C, D, E, H and I
 Heat stable
 Seen in staphylococcal food poisoning
 Increases intestinal peristalsis
 Consuming food products (bakery
goods, custards, potato salad, ice
cream) having preformed toxins
 Self limited (24-48 hours); supportive
therapy
Thursday, October 13, 2016 30

31.  Superantigens
 Staphylococcal enterotoxins and TSST -
1
 Share 3 biologic characteristics
◦ Pyrogenicity
◦ Superantigenicity
◦ Ability to enhance lethal effects of minute
amounts of endotoxin in rabbits upto 100000
fold
 Superantigenicity: ability to stimulate
proliferation of T-lymphocytes without
regard for their antigenic specificitiesThursday, October 13, 2016 31

32. Pathogenesis
 Colonisation
 Introduction into tissues
◦ Minor abrasion
◦ Instrumentation
 Invasion
◦ Serine protease, hyaluronidase, lipase,
etc
 Evasion of host defences
 Metastatic spread
◦ Hematogenous
Thursday, October 13, 2016 32

33. Clinical manifestations
 Skin and soft tissue infection
◦ Folliculitis
◦ Furuncle (boil)
◦ Carbuncle
◦ Impetigo
◦ Hidradenitis suppurativa
◦ Botryomyocosis
Thursday, October 13, 2016 33

34.  Musculoskeletal infection
◦ Septic arthritis – knee, shoulder, hip,
phalanges
◦ Osteomyelitis – children: long bones,
adults: vertebrae
◦ Pyomyositis – skeletal muscle infection –
in tropics and HIV infected people
◦ Abscess – psoas abscess and epidural
abscess
Thursday, October 13, 2016 34

35.  Respiratory tract infections
◦ VAP in adults
◦ Septic pulmonary emboli
◦ Post viral pneumonia (influenza)
◦ Empyema and Pneumothorax
◦ Pneumomatocele – neonates : MCC : SA
Thursday, October 13, 2016 35

36.  Bacteremia and complications
 Sepsis, septic shock
 CLABSI
 Metastatic foci of infection
involving kidney, joints, bone and
lung
 Infective endocarditis
◦ Native valve
◦ prosthetic
◦ i/v drug assoc endocarditis
Thursday, October 13, 2016 36

37.  UTI
◦ SA UTI and pyelonephritis occur
secondary to bacteremia
◦ Following instrumentation – rare
 Toxin mediated
◦ TSS
◦ Food poisoning
◦ SSSS
Thursday, October 13, 2016 37

38. Laboratory diagnosis
 Sample collection
 Direct smear Microscopy
 Culture
 Biochemicals
 Typing of SA
 ABST
Thursday, October 13, 2016 38

39.  Sample collection
Infection Specimen
Suppurative lesion Pus, wound swab
Respiratory infection Sputum
UTI Mid stream urine
PUO, Bacteremia Blood
Food poisoning Feces, Vomitus, food
Carriers Nasal and perianal swab
Thursday, October 13, 2016 39

40.  Direct smear Microscopy
 SA appear as GPC 0.5 to 1.5 microns
 Singly, in pairs, short chains or clusters
 Both within and outside PMNs
 Reports should include quantitation of cell
types and microorganisms
 Eg. Many PMNs, moderate GPC seen
 If Gram stain more typical, report “ GPC
resembling Staphylococci seen”
 Note: Gm stain cant be used to differentiate
staphylococci from micrococci and related
genera or from planococci
Thursday, October 13, 2016 40

41. Thursday, October 13, 2016 41

42.  Culture
 Overnight incubation at 37 deg C
 NA
 1-3 mm, circular, smooth, convex,
opaque, easily emulsifiable, butyrous
 Most strains produce non diffusible
Golden yellow pigment
 Pigmentation can be enhanced – Tween
Agar
 NA slope
◦ Confluent growth, Oil paint appearance
Thursday, October 13, 2016 42

43.  SBA
◦ Colonies similar to those on NA; in
addition surrounded by a narrow zone of
beta-hemolysis
 Liquid medium –
◦ Uniform turbidity
 MA
◦ Small pink colonies due to Lactose
fermentation
Thursday, October 13, 2016 43

44.  Selective media
 Eg. Swabs from carriers, feces
 MSA – NA + 7.5% NaCl + phenol red
 Salt milk agar – NA + 6.5% NaCl +
10% skimmed milk
 Ludlam’s medium – Lithium chloride
and tellurite
Thursday, October 13, 2016 44

45. Thursday, October 13, 2016 45

46.  Biochemicals
 Catalase : +ve
◦ In 3% H2O2
◦ Immediate and vigorous bubbling
◦ Conversion of H2O2 to water and O2 gas
◦ Ideally, not done from blood containing
media
Thursday, October 13, 2016 46

47. Hugh and Leifson O-F test
Staph – fermentative
Micrococci – oxidative
Thursday, October 13, 2016 47

48. S.aureus v/s CONS
Thursday, October 13, 2016 48

49. Coagulase test
Tube test Slide test
 Detects free coagulase
 Reacts with CRF
 Colony of SA is emulsified in 1 ml of
diluted plasma (1:6) in a test tube
and incubated in water bath at 37C
for 4 hrs
 Positive – clot formation
 Negative – RT overnight
 False positive – Citrated plasma
(Pseudomonas, Enterococcus)
utilise citrate
 Can be done with Blood culture
pellets
 Rarely, S.intermidius, S.hyicus,
S.delphini, S.schleiferi may be tube
positive
 Detects clumping factor
(bound coagulase)
 If Neg, do tube test
 Not done from media
with High salt content –
autoagglutinate
 Note: S.lugdunensis,
S.Schleiferi may be
positive
Thursday, October 13, 2016 49

50. Thursday, October 13, 2016 50

51.  DNAse test
 On DNA agar, a clear halo is produced
surrounding the colonies of SA due to its
ability to digest DNA
 Phosphatase test
 Positive for SA, S.epidermidis, S.xylosus
 Organism is inoculated on
phenolphthalein diphosphate containing
media and later colonies are exposed to
ammonia vapour
 Pink colonies
Thursday, October 13, 2016 51

52. Typing of S.aureus
 Epidemiological purpose to trace
source of infection
 Useful in outbreaks like food poisoning
in community
◦ Phenotypic methods – bacteriophage
typing, antibiogram typing
◦ Genotypic methods – PCR-RFLP,
ribotyping, PFGE and sequence based
typing
Thursday, October 13, 2016 52

53.  Bacteriophage typing
 Differentiated into subsp level
 Method
◦ Test train inoculated as lawn culture on NA
◦ Drops of routine test dose of known set of
different phages are spot inoculated
◦ Zone of lysis will be produced in those areas
where test strain is susceptible to phages
applied
◦ If strain lysed by phages 29, 52A, 79, but not
other phages; it is designated as phage type
29/52A/79
◦ National Reference Centre: MAMC, New
Delhi Thursday, October 13, 2016 53

54. Thursday, October 13, 2016 54

55. Antibiotic Sensitivity Testing
 Production of beta lactamase enzyme
◦ Plasmid coded
◦ Produced by >90% SA strains
◦ Overcome by addition of Beta lactamase
inhibitor eg. Clavulanic acid, sulbactam
 Alternation of PBP
◦ Shown by MRSA strains
◦ Chromosomally mediated
◦ mec A gene present
◦ Alters PBP to PBP-2a
◦ CA-MRSA and HA-MRSA
Thursday, October 13, 2016 55

56.  Significance of MRSA
 Mechanism of MRSA
 Which antimicrobials are ineffective on
MRSA?
◦ Penicillins, Cephalosporins, Monobactams, BL-
BLI combinations, Carbapenems.
 Molecular methods “Gold standard” for
detection
Thursday, October 13, 2016 56

57. Methods of ABST
 Divided into types based on the principle
applied to each system
 Advantage of Agar dilution: Multiple isolates
can be tested simultaneously
 Advantage of Broth dilution: Same tubes can
be used for MBC detection
Diffusion method Dilution method Diffusion & Dilution
Stokes method Agar Dilution E-test method
Kirby Bauer method Broth Dilution
Thursday, October 13, 2016 57

58.  Minimum Inhibitory Concentration
(MIC)
 Lowest concentration of an
antimicrobial agent that prevents
visible growth of a microorganism in
an agar or broth dilution susceptibility
test
Thursday, October 13, 2016 58

59. Dilution methods (contd.)
 Either broth / agar dilution methods
used
 Series of tubes / plates prepared with
broth or agar medium
 Various concentrations of antimicrobial
agents added
 Tubes / plates inoculated with std.
suspension of test organism
 Incubation at 350 C
 Tests examined and MIC determined
Thursday, October 13, 2016 59

60. Detection of MRSA
 CLSI recommends 3 screening tests
for detection of MRSA (Oxacillin
resistance)
◦ Oxacillin Resistance - Agar dilution
◦ Broth microdilution
◦ Disc diffusion
Thursday, October 13, 2016 60

61. Screen test Oxacillin Resistance mecA mediated Oxacillin resistance
(Using Cefoxitin)
Organism group S.aureus S.aureus S.aureus
Test Method Agar dilution Disk diffusion Broth microdilution
Medium MHA with 4% NaCl MHA CAMHB
Antimicrobial
Concentration
6µg/mL Oxacillin 30 µg Cefoxitin disk 4µg/mL Cefoxitin
Inoculum Direct colony
suspension to obtain
0.5McFarland turbidity
Using 1 - µL loop that
was dipped in the
suspension, spot an
area 10-15 mm in
diameter. Alternatively,
using a swab dipped
in the suspension and
expressed, spot a
similar area or streak
an entire quadrant
Standard disk diffusion
procedure
1. Inoculum preparation
by Direct colony
suspension
2. Inoculation of plates
3. Application of discs
4. Reading plates &
interpretation
Standard broth
microdilution
procedure
Incubation
conditions
33 to 35 0C; ambient air. (Testing at temperatures
above 350C may not detect MRSA)
Incubation length 24 hours; read with
transmitted light
24 hours 24 hours
Thursday, October 13, 2016 61

62. Screen test Oxacillin Resistance mecA mediated Oxacillin resistance (Using Cefoxitin)
Organism group S.aureus S.aureus S.aureus
Test Method Agar dilution Disk diffusion Broth microdilution
Results Examine carefully with
transmitted light for >1 colony
or light film of growth.
>1 colony = Oxacillin
resistant
≤ 21 mm = mecA positive
≥ 22 mm = mecA
negative
>4µg/mL = mecA positive
≤ 4 µg/mL = mecA
negative
Further testing and
reporting
Oxacillin resistant
staphylococci are
resistant to all β-
lactam agents; other
β-lactam agents
should be reported as
resistant or should not
be reported.
Cefoxitin is used as a surrogate for mecA-
mediated Oxacillin resistance.
Isolates that test as mecA positive should be
reported as Oxacillin (not cefoxitin) resistant;
other β-lactam agents, except those with anti
MRSA activity, should be reported as resistant
or should not be reported.
QC recommendations-
Routine
S.aureus ATCC 29213 –
Susceptible (with each
test day)
S.aureus ATCC 25923 –
mecA negative (Cefoxitin
zone 23-29mm )
S.aureus ATCC 29213 –
mecA negative (Cefoxitin
MIC 1-4 µg/mL)
QC recommendations –
Lot / Shipment
S.aureus ATCC 43300 –
Resistant
S.aureus ATCC 43300 –
mecA positive (zone ≤ 21
mm)
S.aureus ATCC 43300 –
mecA positive (MIC >4
µg/mL)
Thursday, October 13, 2016 62

63.  QC recommendation – Routine
◦ Test negative (susceptible) QC strain:
 With each new lot/shipment of testing materials
 Weekly if the test is performed at least once a week
and criteria for converting from daily to weekly QC
testing have been met
 Daily if the test is performed less than once per week
and /or if criteria for converting from daily to weekly
QC testing have not been met
 QC recommendation – Lot/shipment
◦ Test positive (resistant) QC strain at minimum
with each new lot/shipment of testing
materials.
Thursday, October 13, 2016 63

64. Chromogenic medium used
 HiCrome MeReSa Agar (HiMedia, Mumbai)
a. Ingredients: Casein enzymic hydrolysate, Yeast
extract, Beef extract, Agar, Sodium chloride
Sodium pyruvate Chromogenic mixture
b. Final pH ( at 25°C) 7.0±0.2
c. MeReSa Selective Supplement (FD229) &
Cefoxitin Supplement (FD259) is added
d. Incubation at 30-35°C for 18-48 hours
e. Result: MRSA gives bluish green coloured
colonies
 Study : 3.3% (10/300) were MRSA carriers.
All isolates Cefoxitin resistant.
Thursday, October 13, 2016 64

65. Chromogenic media (contd.)
 CHROMagar MRSA (Paris, France)
 BBL CHROM agar (BD, Diagnostics,
USA)
 MRSASelect (BioRad, USA)
 BioSynth AG (Switzerland)
Thursday, October 13, 2016 65

66. Control Measures
 Proper Hand washing
 Screening of MRSA carriers
 Treatment of Carriers
◦ Topical 2% mupirocin – nasal carrier
◦ Chlorhexidine – skin carriers
 Stoppage of misuse of AMA
 Bundling -
Thursday, October 13, 2016 66

67. Thank you!!!
Thursday, October 13, 2016 67