Staphylococcus

Microbiology of Staphylococcus - Basic

1. Staphylococcus
Anup Muni Bajracharya

2. Introduction
Staphylococcus
➢Gram positive cocci, belongs to Family Micrococcaceae.
➢Aerobic , some facultative anaerobes.
➢Arrangement in clusters ( like grapes), pairs or tetrads,
➢catalase positive.
➢S. aureusis the most important human pathogen, others
human pathogens are CONS (coagulase negative Staphylococci)
which include S. saprophyticus, S. epidermidis, S. hominisetc.
➢Staphylococcus are capable of acquiring resistance to
antibiotics,
➢Can cause serious clinical and epidemiological problems.

3. Human Infections caused by Staphylococcus
❖S. aureuscause:
➢Skin infections like impetigo (rashes ,red
sores), folliculitis(infection in hair follicles),
wound infection.
➢Systemic infections like bacterimia ( viable
bacterial cells in blood), pneumonia, meningitis,
deep-seated abscess.
➢Toxin mediated infectionslike food poisoning,
toxic shock syndrome.
❖S. epidermidiscause :
➢Opportunistic infections like intravenous
catheter infections, CSF shunt infections.
❖S.saprophyticuscause:
➢UTIs.

4. Topics
•Morphology
•Culture
•Biochemical reactions
•Antigenic structure
•Virulence factors
•Pathogenesis ( clinical syndromes)
•Lab diagnosis
•Treatment , prevention and control
•CONS and MRSA.

5. Staphylococcus aureus
MORPHOLOGY :
➢Gm +vecocci, 1µm in diameter.
➢Non motile, Non sporing,
➢Arranged in grape like structure when grown in solid media (due to
incomplete separation of daughter cells during successive divisions of
bacteria)
➢Short chains in liquid media.
➢Facultative anaerobes
➢Grows in large, round , opaque colonies
➢Withstand high salt, extreme pH
➢Normal flora of skin, nasal area.

6. Culture
➢Staphylococcus grows in wide range of media ; Mannitolsalt agar,
Nutrient agar (NA) , Blood agar (BA), Macconkey agar etc. Primary
isolation on NA and BA.
➢On NA, S. aureusgives round, convex, smooth glistening , golden yellow
colonies( believed to be lipoprotein allied to carotene).
➢On BA, S. aureusgives a clear zone of hemolysis(beta-hemolysis), well
marked on sheep or rabbit BA.
➢Other species of Staph do not produce hemolysis.
➢On Macconkey agar, S. aureusproduce small pink colonies due to
fermentation of lactose.
➢On Mannitolsalt agar, S. aureusferment mannitolwith acid production,
gives yellow zone formation around colonies.
➢On liquid media , S. aureusgives turbidity & no production of pigment.

8. Biochemical reactions
➢S. aureusgives catalase positive
➢Coagulase and phosphatase positive but oxidase negative.(used for
differentiation).
➢S. aureusferments mannitol, sucrose, maltose under aerobic conidtion.
➢MR positive , VP positive but indolenegative.

9. Biochemical tests

10. Cell wall components and antigenic structure

11. Virulence factors
➢S. aureusproduce different virulence factors:
Cell wall associated
Polymers and protein
❖Capsular
polysachharide
❖Teichoicacid
❖Protein A
Enzymes
❖Coagulase
❖Catalase
❖Hyaluronidase
❖Penicillinase
❖Nuclease
❖Lipase
Toxins
❖Toxic shock syndrome
toxin
❖Enterotoxin
❖Exfoliativetoxin
❖Leukocidintoxin
❖Hemolysin

13. Pathogenesis
➢Cause localisedlesions
➢First multiply in tissues, produce toxins and stimulate inflammation.
➢Adhere to damagedskin.
➢Evade defense mechanism of host.
➢Cause tissue damage and form abscesses, produce extracellular enzymes
and exotoxins.
Host immunity:
▪No life long immunity.
▪Repeated infections occur in susceptible host.

14. Clinical syndrome:
➢2 types: inflammatory and toxin mediated staphylococcal disease.
INFLAMMATORY DISEASES :
➢Impetigo, folliculitis, furuncle ,carbuncle, surgical wound
infection,postpartumbreast infection.
➢Bacterimia( bacteria found in blood) and septicemia (bacterimia+clinical
symptoms)
➢Endocarditis.
➢Osteomyelitisand arthritis.
➢Deep seated abscess in any organ after bacterimia.

15. Toxin mediated Staphylococcal disease:
1.Staphylococcal food poisoning :
➢Caused by enterotoxin.
➢Milk , milk products, meat fish when kept at room temp after cooking,
contaminants multiply and produce toxins.
➢Virulent when release of interoleukins( IL-1 and IL-2).
➢Sudden symptoms after 2-6 hour of ingestion
➢Nausea, vomiting, abdominal cramps, watery or bloody diarhhea.

16. 2. Staphylococcal toxic shock syndrome (TSS):
➢Caused by Toxin shock syndrome toxin ( TSST).
➢Release of large amtof interleukins IL-1 and IL-2.
➢Life threatningcondition.
➢Fever, vomiting, hypertension, myalgia(muscle pain) mucosal
hyperemia, erythematous rash ( redness due to increased blood
flow).
3. Staphylococcal scaled skin syndrome (SSSS):
➢Caused by exfoliativetoxin
➢common in infants and children.
➢Outer layer of skin of epidermis is separated from underlying
tissue.
➢Appears as extensive bullae( rupture and leave behind red ,
tender skin).

17. Lab Diagnosis
➢Sample collection
➢Direct smear microscopy
➢Culture
➢Biochemical tests
➢Typing of S. aureus
➢Antibiotic susceptibility test (AST)

18. Variousspecimens collected in
staphylococcal infections
Specimen Condition
Pus Supurrativelesions
Sputum Respiratory infections
Blood Bacterimia
Feces vomitus Food poisoning
Urine UTIs

19. Microscopy
➢Gmpositive cocciin clusters and pus cells in Gram stained smear
of pus
➢Not adequate to differentiate.
Culture
➢On NA, large, circular, smooth , glistening colonies, golden yellow
pigments.
➢On BA, zone of beta-hemolysis ( S. aureus)
➢Heavily contaminated sources inoculated in selective media like
ManitolSalt Agar.( S. aureusferments mannitol, gives yellow
color.
Identification of S. aureus
➢Coagulase positive
➢Phosphatase positive
➢Novobiocinsensitive
➢PolymyxinB sensitive

20. Biochemical test
•The main test being coagulase test (tube and slide coagulase)
Tube coagulsetest
•To detect free coagulase
•0.1 ml of overnight broth culture+ 0.5ml undiluted human or
rabbit plasma
•Incubation on water bath at 37°C for 3-6 hours.
•In +vetest, plasma coagulates and doesn’t flow.

21. Slide coagulase test
➢To detect bound coagulase or clumping factor.
➢Mix suspension of bacteria with loopfulof rabbit plasma.
➢In +vetest, clumping occurs.

22. AST(antibiotic sensitivity test)
Novobiocinsenstivity:
➢ZOI > 16mm.
➢differentiation of S.aureusfrom others.
PolymyxinB resistance :
➢Disc diffusion
➢PolymyxinB disc on overnight culture of staph on MHA.

23. Treatment
➢Spontaneous or surgical drainage of pus and debridement.
➢Systemic antibiotics necessary for deep seated and systemic
infections
➢Benzyl penicillin for penicillin resistant strains of S. aureus.
➢Erythromycin, vancomycin, first generation cephalosporins
recommended.

24. Penicillin resistance in Staphylococci
➢Increasing since 1945.
➢80% or more strains are resistant to penicillin.
3 types:
1.Plasma mediated resitance( production of penicillinase
enzyme which is mediated by plasma)
2.Chromosomal mediated resistance( reduces affinity of
penicillin binding proteins to beta lactam antibiotics)
3.Tolerance to penicillin.

25. Prevention and control
➢No effective immunization with toxoids or bacterial vaccines.
➢Maintaining cleanliness
➢Frequent hand washing, and aseptic management of lesions.
➢Creams containing neomycin and bacitracin prevent recurrent
infection
➢Topical application of antimicrobial agents prevent
dissemination of infection from abscess.

26. Coagulase negative Staphylococci
(CONS)
S. epididermis
▪White colonies on BA
▪Catalsepositve, coagulas–ve
▪Doesn’t ferment mannitol.
▪Highly antibiotic resistant.
▪produces slime(virulence factor)
▪Hospital acquired infection.
▪Adhere to intravenous plastic
catheters.
▪Cause endocarditis in patients with
prsotheticvalves.
▪Intravenous catheter infection
▪CSF shunt infection.
▪Sensitive to novobiocin.
S. saprophyticus
•similar
•Similar
•Cause UTIs in sexually active women.
•Adheres to epithelial cells lining the
urogenital tract.
•Dysuria, pyuria, hematuria.
•Prostatitis in elderly men.
•Resistant to novobiocin
(distinguishing character)

27. TEST S.Aureus S. epidermidis S. saprophyticus
1.Coagulase + - -
2. Clumpingfactor + - -
3. Heat stable
nuclease
+ - -
4. Urease variable - +
5. B-galactosidase - - +
6.Polymyxin B Resistant Resistant Sensitive
7. Novobiocin Sensitive Sensitive Resistant
8. Acid from
mannitol
+ - -
Differences bteweenS. aureus, S. epidermidis,S. saprophyticus.

28. Other CONS
➢S. haemolyticus( cause bacterimia, endocarditis, UTIs, wound
infection)
➢S. saccharolyticuscause endocarditis
➢S. hominiscause bacterimiain cancer patients.