Staphylococcus aureus basic exercise

Staphylococcus aureus Basic Identification in Applied Microbiology

Exercise

Dr.T.V.Rao MD

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Patient presented with

• A 30 years male patient presented with an abscess left mandibular region since 3 days, with onset of fever.

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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What are bacteriological agents can produce this pyogenic

condition ?

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Most important bacteria are

Most abscesses are due to infection by

Staphylococcus (as in the boil abscess), but other bacteria are sometimes associated Streptococci, Hemophilus spp. E.Coli Klebsiella spp Pseudomonas spp and many others.

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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How you proceed to identify the etiological agent

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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How to make a Definitive Diagnosis

• To make a definitive diagnosis and to confirm that Staphylococcus is the bacteria causing the infection, Microscopic observation of the given specimen and a culture can be done. Once a bacteria is identified on a culture, the pattern on sensitivities to antibiotics can help to tell whether or not it is actually MRSA, routine staph aureus, or another bacteria.

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Gram staining showing Gram positive Cocci in clusters

– Probably Staphylococcus spp

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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What are Staphylococci: Gram positive cocci ( from Greek

staphyle, means bunch of grapes ) that occur singly and in pairs, short chains

and irregular grape-like clusters.

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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• Gram-positive cocci, nonmotile, facultative anaerobes

• Cells occur in grapelike clusters because cells division occurs along different planes and the daughter cells remain attached to one another

• Salt-tolerant: allows them to tolerate the salt present on human skin

• Tolerant of desiccation: allows survival on environmental surfaces (fomites)

Structure and Physiology of Staphylococcus

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Staphylocoous appear as

• Gram stained smear showing Gram positive cocci with cluster formation indicates presence of Staphylococci

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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III.

Virulence factors of Staphylococcus aureus

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Staphylococcus aureus grown on Nutrient agar

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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General Cultural Characteristics

• Some Staphylococcus species, most notably S. aureus, produce a hemolysin that completely lyses red blood cells of humans and some other mammals (sheep blood). This is referred to as “beta” hemolysis.

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What are your observation on Blood Agar

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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What are the commonly performed in vitro pathogenicity tests?

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Staphylococcus aureus: is the staphylococcus which has the ability to

clot plasma, which is coagulase positive. More than 80% of

Staphylococcus aureus strains produce beta-lactamases.

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Other S. aureus Characteristics Mannitol fermentation is another useful

characteristic – it is unique to, and consistent among S. aureus strains. Virtually all strains of S. aureus ferment mannitol.

Bright yellow colonies on a yellow background indicates mannitol fermentation on mannitol salt agar.

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Coagulase test for Identification of Staphylococcus

Staphylococcus aureus coagulation of plasma* “Free” staphylocoagulase Fibrinogen + CRF Fibrin

Normal coagulation of plasma

Thrombin Fibrinogen Fibrin (soluble ) (insoluble)

*EDTA Rabbit plasma is preferred for the “free” or tube coagulase test because it contains a large amount of CRF

Slide Coagulase Test

• The most important distinction among staphylococci is whether or not they produce the enzyme coagulase. S. aureus the most common pathogen among the catalase positive gram positive cocci and is differentiated from other staphylococci by the coagulase test. Here the bacterial cells have been suspended in a drop of rabbit plasma. Coagulase bound to the cell wall acts on fibrinogen and causes the clumping of the bacteria (right). Coagulase is an important virulence factor of S. aureus

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Tube Coagulase Test

• The tube coagulase test detects both free and cell bound coagulase of S. aureus. Bacteria are incubated in plasma for 2-4 hours and the tubes turned on their sides as illustrated. Free coagulase acts on prothrombin and fibrinogen in plasma and forms a fibrin clot (left). In manylaboratories staphylococci are simply differentiated as coagulase positive or coagulase negative without speciation

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Mention commonly used Antibiotic agents for Staphylococcus aureus

• Penicillins • However Penicillin

developed early resistance • Other Beta lactam

antibiotics like Ampicillin • Macrolide group • Vancomycin, Linezolid

( Only in MRSA strains ). • Commonly used oral anti

staphylococcal antibiotics include the first-generation cephalosporins like cephalexin and cefadroxil.

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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Plate 1a: Staphylococcus aureus resistant to penicillin (P0.5) only. Note the annular radius of the zone of inhibition of 9.5 mm around the cefoxitin (FOX 10) disc.

What is the importance of Drug resistance in this isolate?

• MRSA is an acronym

for Methicillin Resistant Staphylococcus aureus, a bacteria that has become resistant to many antibiotics, including methicillin, penicillin, amoxicillin, and cephalosporins. It is routinely pronounced M.R.S.A.

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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What are the Other Diseases produced by Staphylococcus aureus

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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–Systemic Diseases • Toxic shock syndrome-TSS toxin is absorbed into the

blood and causes shock

• Bacteremia-presence of bacteria in the blood

• Endocarditis-occurs when bacteria attack the lining of the heart

• Pneumonia-inflammation of the lungs in which the alveoli and bronchioles become filled with fluid

• Osteomyelitis-inflammation of the bone marrow and the surrounding bone

Other Staphylococcal Diseases

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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How you Prevent Staphylococcal Infections

• Prevention • Staphylococcus is a

important cause of Hospital infection – Hand antisepsis is the most

important measure in preventing nosocomial infections

– Also important is the proper cleansing of wounds and surgical openings, aseptic use of catheters or indwelling needles, an appropriate use of antiseptics

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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• The programme is created for Undergraduate students in Medical

Colleges as a part of Applied Microbiology exercises

Email

doctortvrao@gmail.com

Dr.T.V.Rao MD Travancore Medical College, Kollam Kerala

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