Staphylococcus by nissim

Nissim Ghimire
M.Sc. Medical Microbiologist
Freelance Microbiology Trainer and lecturer.


 Staphylococci are gram positive cocci,
 Occur in grape like clusters,
 First observed by Von Recklinghausen(1871) in human
pyogenic lesions
 Pasteur(1880) obtained liquid culture from pus and
produced abscesses by inoculating them into rabbits
 Sir Alexender Ogston gave it the name
“STAPHYLOCOCCUS”
 In Greek; staphyle - Bunch of grapes
Kokkus - Berry
Introduction & History

 Staphylococci strain from:
 Pyogenic lesions: produce golden yellow colonies (S
.aureus)
 Normal skin: white colonies (S.albus)
 S.citreus: lemon yellow colonies
 Human skin:
 coagulase negative staphylococcus
 S.aureus :lesser extent
 Prefered habitat:anterior nares (40%adults carriers)
 Micrococci :in skin and in environment
 Stomatococcus(s.mucilaginosus):normal human oral flora
Contd…


 Species of Staphylococci found in human skin:
 S.saprophyticus
 S.epidermidis
 S.haemolyticus
 S.hominis
 S.warneri
 S.lugdunensis
 S.simulans
 S.xylosus
Contd…


 Morphology:
 Gram positive cocci
 Diameter:1 µm (approx.)
 Arranged in grapes like clusters
 Non-motile
 Non-spore forming
 Non capsulated (some strains posseses
microscopically visible capsule (young culture))
Staphylococcus aureus

Culture:
Media used :-
i) Non selective media: Nutrient agar,
Blood agar,
MacConkey’s agar.
ii) Selective media: Salt-milk agar,
Ludlam’s medium
Robertson’s cooked meat medium with 10% sodium
chloride
 Temperature range :10°C - 42°C (optimum temp: 37°C)
 pH:7.4-7.6
 Aerobes & facultative anaerobes
Culture characteristics:

Culture
characteristics:
i) On nutrient agar: The
colonies are :
 large circular,
 convex,
 smooth,
 shiny,
 opaque and
 Easily emulsifiable.
 Most strains produce
golden yellow pigments.

ii) On MacConkey’s agar- The colonies are small & pink in
colour. (due to lactose fermentation)
iii) On blood agar- Most strains produce β- haemolytic
colonies. (specially incubated under 20-25%
carbondioxide)
iv) On liquid medium: uniform urbidity is produced

Biochemical reactions:
1.Catalase test- Positive.
2H2O2  O2 + 2H2O


2) Coagulase test-
i) Slide coagulase test- Positive.
ii) Tube coagulase test- Positive.
SLIDE COAGULASE TEST TUBE COAGULASE
TEST

Contd….
3) Reduces nitrate to nitrite.
4) Ferments mannitol anaerobically with acid only.
5) Urea hydrolysis test- Positive.
6) Gelatin liquefaction test- Positive.
7) Produces Lipase.
8) Produces Phosphatase.
9) Produces Thermostable nuclease.
10) MR & VP positive
11) Indole negative
12) Bacitracin resistant
13)Grow on agar that contains peptone
15)Some are ß-hemolytic

 Staphylococci are among the more resistant non-spore
forming bacteria
 Remain viable for 3-6 months (have been isolated from dried
pus after 2-3 months
 May withstand 60°C for 30 minutes (thermal death
time:62°C for 30 minutes)
 some Staphylococci require heating at 80°C for 1 hour to
be killed
 Heat resistant strains have ability to grow in higher
temperature even at 45°C
 Resist 1 % phenol for 15 mins.
 Mercury peroxide 1% solution can kill them in 10 mins.
Resistance :

Penicillin resistance
 3 types:
 Production of Beta lactamase
 Alteration in penicillin binding protein PBP2a
 Development of tolerance to penicillin
 Bacterium is only inhibited but not killed

Contd…
Production of beta lactamase:(penicillinase)
 Inactivates penicillin
 Staphylococci produces 4 types of penicillinases(A-D)
 Hospital strains:type A penicillinase
 Penicillinase:
 inducible enzyme
 Production usually controlled by plasmids,which are
transmitted by transduction or conjugation
 Same plasmid: may carry genes for resistance to a range
of other antibiotics and heavy metals

 Alteration in penicillin binding protein(PBP2a) &
changes in bacterial surface receptors:
 reduces binding of beta lactam antibiotics to cells
 Normally chromosomal in nature
 Expressed more at 30°C than at 37°C
 Also extends to cover beta lactamase-resistant
penicillins such as methicillin and cloxacillins (MRSA)
 EMRSA:’epidemic methicillin-resistant Staphylococcus
aureus’
 erythromycins,tetracyclines,aminoglycosides and heavy
metals
Contd…

PATHOGENICITY:
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
Pathogenicity and virulence

Virulence factors:
These include
A) Cell associated factors
B) Extracellular factors
Contd…

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


 Protein A:present in cell wall of most S.aureus
Strains
 Chemotactic
 Anti-phagocytic:
 Binds to Fc part of IgG
 Blocks phagocytosis
 Anti-complementary
 Induces platelet damage and hypersensitivity

B) EXTRACELLULAR FACTORS
a) Enzymes:
1. Free coagulase
2. Catalase
3. Lipase(infect skin and subcuaneous tissues)
4. Hyaluronidase(hydrolyse hyaluronic acid in
connective tissues)
5. DNAase
6. Thermonuclease
7. Staphylokinase (Fibrinolysin)
8. Phosphatase

Contd…
b) Toxins:
1.Cytolytic toxins(membrane active substance)
(affects RBC and WBC)
i) Haemolysins
 Alpha haemolysin(paradoxic):toxic to
macrophages,lysosomes,muscle tissues,renal cortex and
circulatory system
 Beta haemolysin (sphingomyelinase)
 Shows hot-cold phenomena
 Gamma haemolysin
 Delta haemolysin:
 detergent like effects on cell membreane of
erythrocytes,leucocytes,macrophages and platelets
ii) Leucocidin (Panton-Valentine toxin)


2. Enterotoxin
3. Toxic shock syndrome toxin (TSST)
4. Exfoliative (epidermolytic toxin)


Staphylococcal diseases
Diseases produced by Staphylococcus aureus:2 groups
A) Infections
B) Intoxications

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

Common Staphylococcal infections:
1) Skin and soft tissue: Folliculitis, furuncle (boil), carbuncle,
styes, abscess, wound infections, impetigo, paronychia and
less often cellulitis.
Folliculitis
Furuncle (boil)
Folliculitis

Impetigo Paronychia
Wound infection Cellulitis

2) Musculoskeletal: Osteomyelitis, arthritis, bursitis,
pyomyositis.
osteomyelitis
3) Respiratory: Tonsillitis, pharyngitis, sinusitis, otitis,
bronchopneumonia, lung abscess, empyema, rarely
pneumonia.

4) Central nervous system: Abscess, meningitis, intracranial
thrombophlebitis.
5) Endovascular: Bacteremia, septicemia, pyemia,
endocarditis.
Endocarditis
6) Urinary: Urinary tract infection.

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

1) Food poisoning: (Enterotoxin)
 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.
 The toxin acts directly on the autonomic nervous system to cause the
illness, rather than gut mucosa.


 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.
Contd…

2) Staphylococcal Toxic shock syndrome (STSS):
 STSS is associated with infection of mucosal or sequestered
sites by TSST( formerly known as enterotoxin type F)
producing S.aureus.
 It is fatal multisystem disease presenting with fever,
hypotension, myalgia, vomiting, diarrhoea, mucosal
hyperemia and erythematous rash which desquamates
subsequently.

2 types of STSS known:
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.

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.

Types of SSSS:
Severe form Milder form
In new born - Ritter’s disease - Pemphigus
neonatorum
In older patients - Toxic epidermal - Bullous
necrolysis impetigo

Ritter’s disease Pemphigus neonatorum
Toxic epidermal necrolysis Bullous impetigo

LAB DIAGNOSIS:
1.Specimens: 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.

2.microscopy:
 Direct microscopy with Gram stained smear is useful in
case of pus, where cocci in clusters are seen.
 This is of no value for specimen like sputum where
mixed flora are normally present.

3.Culture:
 Specimens plated in blood agar
 Staphylococcal colonies appear after overnight incubation
 Specimens where staphylococci are expected to be scanty and
outnumbered by other bacteria are inoculated on selective
media
 Salt-milk agar,
 Ludlam’s medium
 Robertson’s cooked meat medium with 10% sodium

1.Gram staining:
Smears are examined from the culture plate and reveals Gram
positive cocci(1μm in diameter) arranged in grape like clusters.
3.Identification:

Differentiation between staphylococci micrococci and
stomatococci
property staphylococcus micrococcus stomatococcus
Anaerobic growth + _ +
Carbohydrate utilization F O F
catalase + + Weak
oxidase _ + _
Bacitracin R S S
lysostaphin S R R
Adherence to agar _ _ +


Differentiation of S.aureus from CONS
Contd…
test S.aureus S.epidermidis S.saprophyticus
Growth on manitol salt agar + _ _
Colonial pigmentation Golden yellow white White
Coagulase test + _ _
DNAase Test + _ _
Hemolysis in blood agar beta _ _
Novobiosin sensitivity S S R


 Staphylococci are primary parasites of human beings and
animals.
 Hospital infections caused by staphylococci deserve
special attention because of their frequency & they are
caused by strains resistant to various antibiotics.
 Staphylococci are the common cause of postoperative
wound infection and other hospital cross infections.
Epidemiology:


 Methicillin-Resistant Staphylococcus Aureus
 Epidemic strains of these; MRSA are usually
resistant to several other antibiotics
 Characteristic of MRSA is its ability to thrive in
presence of penicillin like antibiotics which normally
prevent bacterial growth by inhibiting cell-wall
synthesis.
MRSA


Methicillin-resistant Staphylococcus aureus
(MRSA)
Mechanism
MRSA contains the mecA gene which is responsible for the production of
an altered plasma (cell) membrane-bound enzyme, penicillin-binding
protein 2a (PBP- 2a.) , penicillin-binding protein 2’
Penicillin-binding proteins are enzymes that participate in the production
of a major component of the bacterial cell wall
The altered PBP 2a while able to perform its cell-wall synthesis function,
has a low affinity for and does not bind to beta-lactam antibiotics
mecA, stops β-lactam antibiotics from inactivating the enzymes (trans-peptidases) critical for
cell wall synthesis.


(MRSA)
Thus, the presence of the mecA gene confers resistance to all beta-
lactam antibiotics such as methicillin.
The mec A gene along with several other virulence and/or antibiotic
resistance genes is carried on a movable segment or unit of the
bacterium’s chromosome called the “staphylococcal cassette
chromosome mec ” (SCCmec).
Currently, there are 6 different types; I, II, III, IV, V and VI, and all
of which vary in size
The larger the SCCmec type, the more room there is for resistance
and other genes
Hospital-acquired MRSA strains usually contain types I, II, or III,
while community-associated MRSA strains contain types IV, and V.
Mechanism


(MRSA)


Treatment
(MRSA)
Both CA-MRSA and HA-MRSA are resistant to
traditional anti-staphylococcal beta-lactam antibiotics,
such as cephalexin
CA-MRSA has a greater spectrum of antimicrobial
susceptibility to sulfa drugs (like co-trimoxazole
(trimethoprim/sulfamethoxazole), tetracyclines (like do
xycycline and minocycline)
and clindamycin (for osteomyelitis)
MRSA can be eradicated with a regimen of linezolid,
though treatment protocols vary and serum levels of
antibiotics vary widely person to person and may affect
outcomes


 Linezolid belongs to the newer oxazolidinone
class of antibiotics which has been shown to be
effective against both CA-MRSA and HA-
MRSA.
The effective treatment of MRSA with linezolid has been successful in 87% of
people. Linezolid is more effective in soft tissue infections than vancomycin. This is
compared to eradication of infection in those with MRSA treated with vancomycin.
Treatment with vancomycin is successful in approximately 49% of people
 The Infectious Disease Society of America recommends
vancomycin, linezolid, or clindamycin (if susceptible) for treating
those with MRSA pneumonia.
(MRSA)


(MRSA)
Treatment
Vancomycin remains the drug of choice for treatment of infections caused
by MRSA, although it is intrinsically less active than the antistaphylococcal
penicillins
Combinations of vancomycin with beta-lactam antibiotics may be
synergistic in vivo against MRSA strains, including those with intermediate
susceptibility to vancomycin
Given the increasing prevalence of MRSA in hospitals and in community
settings, alternative approaches are needed for treatment of infections
caused by MRSA.


(MRSA)
Treatment
Vancomycin
and teicoplanin are glycopeptide antibiotics
used to treat MRSA infections
Teicoplanin is a structural congener of
vancomycin that has a similar activity
spectrum but a longer half-life
Because the oral absorption of vancomycin
and teicoplanin is very low, these agents can
be administered intravenously to control
systemic infections.

PREVENTION:
 Isolation & treatment of MRSA
patients.
 Detection of carriers among hospital
staff, their isolation & treatment.
 Avoid indiscriminate usage of
antibiotics.
 Following strict aseptic technique
 Hand washing,oldest simplest and
most effective method of previnting
hospital cross-infection.

Other coagulase positive Staphylococci:
 Staphylococcus aureus subsp. Anaerobius
 S. a. aureus
 S. hyicus
 S. intermedius and
 Staphylococcus schleiferi subsp. coagulans.

Coagulase Negative Staphylococci( CoNS ):
Two species of coagulase negative
Staphylococci can cause human infections-
1. Staphylococcus epidermidis
2. Staphylococcus saprophyticus

S. Epidermidis:
 It is a common cause of stitch abscesses.
 It has predilection for growth on implanted foreign bodies such
as artificial valves, shunts, intravascular catheters and
prosthetic appliances leading to bacteraemia.
 In persons with structural abnormalities of urinary tract, it can
cause cystitis.
 Endocarditis may be caused, particularly in drug addicts.

S.saprophyticus:
 It causes urinary tract infections, mostly in sexually active
young women.
 The infection is symptomatic and may involve the upper
urinary tract also.
 Men are infected much less often.
 It is one of the few frequently isolated CoNS that is resistant
to Novobiocin

Other coagulase negative staphylococci:
 S.haemolyticus
 S. saprophyticus
 S. warneri,
 S.hominis,
 S.epidermidis
 S. caprae and
 S.lugdunensis

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