Experiment 25, 26, and 27 Identification of Staphylococcus Species

DNase agar

Contains an emulsion of DNA, peptides, and methyl green dye. The dye and polymerized DNA form a complex that gives the agar a blue-green color at pH 7.5

Coagulase test

a test in which organisms are mixed with plasma on a slide. if the cells clump together, the culture is coagulase positive. Allows differentiation of S. aureus from other staph strains by the fact that most S. aureus strains produce an enzyme called coagulase.

Coagulase

acts within host tissue to convert fibrinogen to fibrin

Virulent strains of Staph aureus produce coagulase which converts...

Fibrinogen to fibrin

Mannitol Salt Agar

Staphyloccus aureus will have the ability to digest mannitol

Members of the Staphyloccus genus can be distinguished from other Gram-positive cocci by two main properties:

1. the ability to grow on high salt media, and 2. the production of the enzyme catalase.

Mueller-Hinton agar

A common agar medium used for anti-microbial susceptibility testing. In this lab, the M-H agar will be used to test susceptibility to the antibiotic Novobiocin.

Rapid staph test

Based on Staphylococcus aureus producing coagulase and Protein A antigens on surface. Coagulase converts fibrinogen in blood to fibrin in order to produce a clot (for hiding from immune system). Protein A binds to Fc region of IgG molecules. Staph kit contains latex particles coated with fibrinogen and IgG. Positive tests should have agglutination reactions.

Protein A

Binds Fc region of IgG. Prevents opsonization and phagocytosis. Expressed by S. aureus.

What advantages does possessing the enzyme DNase confer on bacteria? How could DNase aid in the virulence of pathogenic bacteria?

By breaking down the possibility of synthesizing any antibodies. It has been shown that DNase helps Group A strep bacteria escape neutrophil extracellular traps (NETs)

Virulent factors of Staph. aureus

Coagulase and Protein A

Another emerging-type of MRSA

Community-acquired MRSA (CA-MRSA), not hospital associated, and is causing serious infections in otherwise healthy people

Emergence of MRSA

Emerged in hospitals and hospital-like settings in the early 1960s, and has become a significant threat to public health

Diseases associated with opportunistic S. epidermidis

Endocarditis, bacteremia, meningitis, UTI, peritonitis

S. aureus characteristics

Ferments MSA completely, tests positive for DNase, Susceptible to Novobiocin, tests positive for coagulase

What two substances are used to coat the latex beads in the rapid Staph test?

Human fibrinogen and IgG

How does coagulase contribute to the virulence of pathogenic S. aureus?

In human host, the action of coagulase enzyme produces clotting of the plasma by converting fibrinogen to fibrin in the immediate vicinity of the bacterium as a means of protection by itself. The fibrin meshwork that is formed by this conversion surrounds the bacterial cells or infected tissues, protecting the organism from non-specific host resistance mechanisms such as phagocytosis and the anti staphylococcal activity of normal serum. This enables the bacterium to persist in the presence of a host immune response, which can lead to the establishment of infection.

S. saprophyticus

No mannitol fermentation, tests negative for DNase, resistant to Novobiocin, tests negative for coagulase

What species does a rapid staph test for?

S. aureus

Give an example of a Staphylococcus that ferments mannitol.

S. aureus and S. xylosus

Staphylococcus has two main groups:

S. aureus and non-S. aureus

Cultures used

S. aureus, S. epidermidis, S. xylosus, S. intermedius, S. saprophyticus

Give an example of a Staphylococcus that does not ferment mannitol.

S. epidermidis

Other non-S. aureus species most clinically significant

S. epidermidis

Describe how you would perform a catalase test. What result would you expect for a Staphylococcus?

Staphylococci produce an enzyme called catalase, which reacts with the toxic oxygen byproduct hydrogen peroxide, breaking it down into water and molecular oxygen. two drops of 3% h2o2 is put on a clean glass slide. a loopful of staph organism (or strep) is put into the drops. if bubbles appear, it is a clear sign that catalase is reacting to the h2o2

S. epidermidis characterisitcs

Weak fermentation of Mannitol, tests negative for DNase, susceptible to Novobiocin, tests negative for coagulase

Staphylococcus species are distinguished from one another based on:

fermentation of mannitol, their susceptibility to the antibiotic Novobiocin, and whether or not they produce a hydrolytic enzyme called DNase, which degrades DNA.

Novobiocin

inhibits DNA gyrase

Staphylococcus can be distinguished from other pathogenic species by:

it's ability to ferment mannitol and by a positive result in a coagulase test

S. aureus associated diseases

leading cause of skin and soft tissue infections (SSTIs), toxic shock syndrome (TSS), and scalded skin syndrome. Also causative agent in pneumonia, meningitis, arthritis, and osteomyelitis

S. epidermidis

lives on skin and mucous membranes, most common skin staphylococcus, and can be opportunistic often infecting IV drug users, newborns, elderly, and those using catheters or other artificial appliances.

Important subclass of S. aureus

methicillin-resistant S. aureus (MRSA), not only resistant to penicillin-derived methicillin, but to other antibiotics as well

Give an example of a Staphylococcus that is DNase-positive

staph aureus

Give an example of a staphylococcus that is DNase-negative

staph epidermidis and staph xylosus

What does a positive coagulase test look like?

visible clots will appear