Download staphylococcus -study material-2012

STAPHYLOCOCCUS 1
STAPHYLOCOCCI
Staphylococci are Gram-positive cocci that occur in grape-like clusters. They are ubiquitous and are the most
common cause of localised suppurative lesions in human beings. Their ability to develop resistance to penicillin
and other antibiotics enhances their importance as a human pathogen, especially in the hospital environment.
The name Staphylococcus (staphyle, in Greek, meaning 'bunch of grapes'; kokkos, meaning a berry) is due to the
typical occurrence of the cocci in grape-like clusters in pus and in cultures.
The genus Staphylococcus is now classified into 32 species and 15 subspecies based on the chemical composition
of their cell wall components and other properties. Besides Staph aureus, three coagulase- negative speciesStaph epidermidis, Staph haemolyticus and Staph saprophyticus-can also cause human disease. Some other
coagulase-negative species such as Staph hominis and Staph capitus are part of the commensal flora of the
human skin. Other species are parasitic on animals.
Morphology: They are spherical cocci, approximately 1 urn in diameter, arranged characteristically in grape- like
clusters Cluster formation is due to cell division occurring in three planes, with daughter cells tending to remain
in close proximity. They may also
be found singly, in pairs and in short chains of three or four cells, especially when examined from liquid culture.
Long chains never occur. They are nonmotile and nonsporing. A few strains possess microscopically visible
capsules, particularly in young cultures. Many apparently noncapsulated strains have small amounts of capsular
material on the surface. They stain readily with aniline dyes and are uniformly Gram positive. Under the
influence of penicillin and certain chemicals, they may change to L forms.
Cultural characteristics: They grow readily on ordinary media within a temperature range of 10 to 42°C, the
optimum being 37 DC, and a pH of 7.4-7.6. They are aerobes and facultative anaerobes. On nutrient agar, after
incubation for 24 hours, the coloniesare large (2-4 mm diameter), circular, convex, smooth, shiny, opaque and
easily emulsifiable. Most strains produce golden yellow pigment, though some may be white, orange or yellow.
The pigment does not diffuse into the medium. Pigment production occurs optimally at 22°C and only in aerobic
cultures. Pigment production is enhanced when 1% glycerol monoacetate or milk is incorporated in the medium.
The pigment is believed to be a lipoprotein allied to carotene. The colonies on blood agar are similar to those on
nutrient agar.Most strains are hemolytic, especially when incubated.under 20-25% carbon dioxide. Hemolysis is
marked on rabbit or sheep blood and weak on horse blood agar. In liquid media, uniform turbidity is produced.
Several selective media have been devised for isolating Staph aureus from specimens such as feces containing
other bacteria. These include media containing 8~ 10 per cent NaCI (salt-milk agar, salt broth), lithium chloride
and tellurite (Ludlam's medium) , and
polymyxin.
Biochemical reactions: They ferment a number of sugars, producing acid but no gas. Sugar fermentation is of
no diagnostic value except for mannitol, which is usually fermented by Staph aureus but not by other species.
They are catalase positive (unlike
streptococci) and usually hydrolyse urea, reduce nitrates to nitrites, liquefy gelatin and are MR and VP positive
but indole negative. Most strains are lipolytic and when grown on media containing egg yolk, produce a dense
opacity. Production of phosphatase
can be demonstrated by culturing on nutrient agar containing phenolphthalein diphosphate. When such a
STAPHYLOCOCCUS 2
culture is exposed to ammonia vapour, colonies assume a bright pink colour due to the presence of free
phenolphthalein.
Staph aureus strains usually exhibit the following
characteristics:
1. coagulase positive;
2. greater biochemical activity, ferment mannite;
3. produce clear hemolysis on blood agar;
4. produce a golden yellow pigment;
5. liquefy gelatin;
6. produce phosphatase;
7. in a medium containing potassium tellurite, reduce tellurite to form black colonies and
8. produce thermostable nucleases which can be demonstrated by the ability of boiled cultures to degrade
DNA in an agar diffusion test
Resistance: Staphylococci are among the more resistant of non sporing bacteria. Dried on threads, they retain
their viability for 3-6 months.
Pathogenicity and virulence: Staphylococci produce two types of diseases-infections arid intoxications. In the
former the cocci gain access to damaged skin, mucosal or tissue sites, colonize by adhering to cells or
extracellular matrix, evade host defence mechanisms,
multiply and cause tissue damage. In intoxications, the disease is caused by the bacterial toxins produced either
in the infected host or preformed in vitro. The virulence factors described include the
following:
Cell Associated Polymers
 The cell wall polysaccharide peptidoglycan confers rigidity and structural integrity to the bacterial cell. It
activates the complement and induces release of inflammatory cytokines.
 Teichoic acid, an antigenic component of the cell wall, facilitates adhesion of the cocci to the host cell surface
and protects them from complement- mediated opsonisation.
 Capsular polysaccharide surrounding the cell wall inhibits opsonisation.
Cell Surface Proteins: Protein A, present on most Staph aureus strains, has many biological properties, including
chemotactic, antiphagocytic and anti- complementary effects. It also induces platelet damage and
hypersensitivity.
Clumping factor, another surface protein, is the 'bound coagulase' which is responsible for the 'slide coagulase'
test. When a saline suspension of Staph aureus is mixed on a slide with a drop of human plasma the cocci are
clumped. The slide coagulase test is routinely used for the identification of Staph aureus isolates. Capsulated
strains may sometimes show a negative test because the clumping factor may be enveloped by the capsular
polysaccharide.
Extracellular enzymes: Coagulase is an enzyme which brings about clotting of human or rabbit plasma. It acts
with a 'coagulase reacting factor' (CRF) present in plasma, binding to prothrombin and converting fibrinogen to
fibrin.Coagulase is an enzyme secreted into the medium.
Lipases: Staphylococci produce a number of lipid hydrolases which help them infect the skin and subcutaneous
tissues.
Hyaluronidase breaks down the connective tissue. Staphylokinase (fibrinolysin), fatty acid modifying enzymes
and proteases help in initiation and spread of infection.
STAPHYLOCOCCUS 3
Nuclease: A heat stable nuclease is a characteristic feature of Staph aureus.
Protein receptors: Staphylococci possess receptors for many mammalian proteins such as fibronectin,
fibrinogen.
Toxins
Cytolytic toxins: Cytolytic toxins are membrane- active substances, consisting of four hemolysins and a
leucocidin. Alpha hemolysin (alpha toxin, lysin) is the more important among them. It is a protein inactivated at
70°C, but reactivated paradoxically at 100°C. Beta hemolysin is a sphingomyelinase, hemolytic sheep celis, but
not for human or rabbit cells. It exhibits a hot-cold phenomenon, the hemolysis being initiated at 37°C, but
becoming evident only after chilling.
Gamma hemolysin is composed of two separate proteins, both of which are necessary for hemolyti
activity.
Delta hemolysin has a detergent-like effect on the eel membranes of erythrocytes, leucocytes, macrophag and
platelets.
Leucocidin is also a two-component toxin, like the gamma lysin, being composed of two componen (S and F).
Such bicomponent, membrane-active toxins as the staphylococcal leucocidin and gamma lysin has been grouped
as synergohymenotropic toxins.
Enterotoxin: This toxin is responsible for the manifestations of staphylococcal food poisoning- nausea, vomiting
and diarrhea 2-6 hours after consuming contaminated food containing preform toxin. The toxin is relatively heat
stable, resisting 100 for 10 to 40 minutes
Toxic shock syndrome toxin (TSST): Toxic shock syndrome (TSS) is a potentially fatal multisystem disease
presenting with fever, hypotension, myalgia, vomiting, diarrhea, mucosal hyperemia and an erythematous rash
which desquamates subsequently. This is associated with infection of mucosal or sequestered sites by TSSTproducing Staph aureus strains usually belonging to bacteriophage group I. TSST type-l (formerly also known as
enterotoxin type F or pyrogenic exotoxin C depending on the concentration ~ the toxin and nature of the
medium.
Exfoliative (epidermolytic) toxin: This toxin, also known as ET or 'exfoliatin', is responsible for the
staphylococcal scalded skin syndrome' (SSSS), an exfoliative skin disease in which the outer layer of the
epidermis gets separated from the underlying tissues.
The severe form of SSSS is known as Ritter's disease in the newborn and toxic epidermal necrolysis in older
patients.
Staphylococcal Diseases
Staphylococcal infections are among the most common of bacterial infections and range from the trivial to the
fatal. Staphylococcal infections are characteristically localised pyogenic lesions, in contrast to the spreading
nature of streptococcal infections. Cornmon staphylococcal infections are as follows:
Skin and soft tissue: Folliculitis, furuncle (boil), abscess (particularly breast abscess), wound infection, carbuncle,
impetigo, paronychia, less often cellulitis.
Musculoskeletal: Osteomyelitis, arthritis, bursitis, pyomyositis
Respiratory: Tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess, empyema, rarely
pneumonia
Central nervous system: Abscess, meningitis,
intracranial thrombophlebitis ,
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Endovascular: Bacteremia, septicemia, pyemia, endocarditis.
Urinary: Staphylococci are uncommon in routine urinary tract infections, though they do cause infection in
association with local instrumentation, implants or diabetes. Urinary isolates of staphylococci are to be
considered significant even with low colony counts, as they may be related to bacteremia.
Bacteriophage Typing Staphylococci may be typed, based on their susceptibility to bacteriophages. An
internationally accepted set of phages is used for typing. Staphylococcal phage typing is done by a pattern
method. The strain to be typed is inoculated on a plate of nutrient agar to form a lawn culture. After drying, the
phages are applied over marked squares in a fixed dose (routine test dose). After overnight incubation, the
culture will be observed to be lysed by some phages but not by others. The phage type of the strain is expressed
by the designations of all the phages that lyse it. Thus, if a strain is lysed only by phages 52, 79 and 80, it is called
phage type 52/79/80. Phage typing is of great importance in epidemiological studies of staphylococcal infections.
International basic set of phages for typing Staph aureus of human origin
Epidemiology: Staphylococci are primary parasites of human beings and animals, colonising the skin, skin glands
and mucous membranes. The most common sources of infection are human patients and carriers, animals and
inanimate objects being less important. Hospital infections by staphylococci deserve special attention because
of their frequency and because they are caused by strains resistant to various antibiotics. Staphylococci are a
common cause of postoperative wound infection and other hospital cross-infections.
Measures for the control of staphylococcal infection
in hospitals include:
 isolation of patients with open staphylococcal lesions;
Group I- 29, 52, 52A, 79, 80
 detection of staphylococcal lesions among surgeons, nurses
Group II -3A, 3C, 55, 71
and other hospital staff and keeping them away from work till the
Group III- 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85
lesions are healed;
Group IV  strict aseptic techniques in theatres; the oldest, simplest and
Group V -94,96
the most effective method of checking hospital cross-infection is
Not allocated-81,95
hand washing, which unfortunately is often neglected.
Laboratory diagnosis: The specimens to be collected depend on the type of lesion (for example, pus from
suppurative lesions, sputum from respiratory infections). In cases of food poisoning, feces and the remains of
suspected food should be collected. For the detection of carriers, the usual specimen is the nasal
swab. Swabs from the perineum, pieces of hair and umbilical stump
may be necessary in special situations.
Direct microscopy with Gram-stained smears is useful in the case of pus, where cocci in clusters ma_
be seen. This is of no value for specimens like sputum where mixed bacterial flora are normally
present.
Diagnosis may readily be made by culture.
The coagulase test can be done using two methods: tube and slide. The tube coagulase test detects free
coagulase.
About 0.1 ml of a young broth culture or agar culture suspension of the isolate is added to about
0.5 ml of human or rabbit plasma in a narrow test tube. EDTA, oxalate or heparin may be used as
the anticoagulant for preparing the plasma.Positive and negative controls are also set up. The
tubes are incubated in a water bath at 37 DC for 3-6 hours. If positive, the plasma clots and does
not flow when the tube is tilted. Continued incubation is not recommended as the clot may get lysed by the
STAPHYLOCOCCUS 5
fibrinogen formed by some strains.
For the slide test, the isolate is emulsified in a drop of saline on a slide. After checking for absence of
autoagglutination, a drop of human or rabbit plama is added to the emulsion and mixed. Prompt clumping of the
cocci indicates a positive test. Positive and negative controls also are set up.
Antibiotic sensitivity tests should be performed as a guide to treatment. Bacteriophage typing may be done if
the
information is desired for epidemiological purposes. Other typing methods include antibiogram pattern, plasmid
profile, DNA fingerprinting, ribotyping and PCR-based analysis for genetic pleomorphism.
Treatment As drug resistance is so common among staphylococci, the appropriate antibiotic should be chosen
based on antibiotic sensitivity tests. Benzyl penicillin is the most effective antibiotic, if the strain is sensitive. For
life-threatening staphylococcal infections, vancomycin is the drug of choice.
Coagulase-negative Staphylococci Coagulase-negative staphylococci constitute a major component of the
normal flora of the human body. Some species of coagulase-negative staphylococci can produce human
infections-Staph epidermidis, Staph haemolyticus and Staph saprophyticus.
MICROCOCCI These are Gram - positive cocci which occur mostly in pairs, tetrads or irregular clusters. They are
catalase and oxidase positive. They are aerobic with strictly respiratory metabolism. They are parasitic 0
mammalian skin and are ordinarily nonpathogenic, They resemble staphylococci but in stained smears the cells
are generally larger and more Gram variable than staphylococci. In cultures they form smaller colonies.
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