Staphylococcus aureus - Laboratory diagnosis and prevention

LABORATORY DIAGNOSIS:

1.      Specimen collection

• Pus from pyogenic lesions/ wound/ burns.

• blood from septicaemia.

• Cerebrospinal fluid from meningitis.

• sputum from respiratory infection

• suspected food, vomit or faeces from food poisoning.

• Mid-stream urine in urinary tract infection.

• Anterior nasal swab from suspected carriers.

2.      Diagnosis

a)      Direct microscopic examination

1. Gram stained smears

·         Gram positive. cocci (spherical), predominantly in grape-like clusters - single cell or pairs or in short chain also appear

·         Is of no use in specimens like sputum, where mixed cultures are seen

2. Culture and isolation

·         Specimens are cultured on Blood agar plate and are incubated at 37 °C for 24 hours. S. aureus forms β-hemolysis /clear zone around the colonies.

(S. epidermidis & S. saprophyticus - -no hemolysis of red blood cells)

·         On nutrient agar Staphylococcus aureus colonies are: large, circular, smooth , shiny surface and are pigmented (golden-yellow).  

·         On mannitol salt agar,

- It is a differential medium for mannitol fermenters.

-Staphylococcus aureus produce yellow colonies - Other Staphylococci produce small pink or red colonies with no colour change to the medium (non-mannitol fermenter)

·         Selective media used if Staphylococci are expected to be few in  number in the sample or if other bacteria predominate

·         Eg., swabs from carriers/ feces in food poisoning cases)-  Ludlam’s/ salt-milk agar/ Robertson’s cooked meat medium containing 10% sodium chloride

 

3.   3. Biochemical tests

·         Catalase, Phosphatase, Indole production, MR, VP, Urea hydrolysis

·         Coagulase

      Catalase test:  Is used to differentiate between staphylococci (catalase +ve) and streptococci (catalase –ve).

       DNase TEST

• enables the detection of DNase that depolymerize DNA.

•A zone of clearing around the spot or streak indicates DNase activity.

   Coagulase test is used to differentiate Staphylococcus aureus from coagulase-negative staphylococci.

     -Slide coagulase test (bound coagulase)-

Procedure

-Place a drop of sterile water/saline on a slide and emulsify a colony – check for autoagglutination.

-Add a drop of human/rabbit plasma to the suspension

- Observe for prompt clumping/agglutination which indicates Positive test for S. aureus. If, no agglutination, test is negative (other staphylococcci)

 -Tube Coagulase Test (free coagulase)- clot formation when bacterial cells are incubated with plasma.

Procedure

-Inoculate human/rabbit plasma with organism and incubate at 35-37⁰C

-Observe at 30 minutes for the presence of a clot

-Continue for up to 3-6 hours, if needed.

-Observe for plasma clots and it doesn’t flow if the tube is inverted

 4. Novobiocin Susceptibility Test

This test is used to differentiate coagulase-negative staphylococci, especially in urine isolates. 

    S.aureus is susceptible to Novobiocin whereas CONS S. saprophyticus is  resistant to the antibiotic novobiocin. 

 5.      API Test

API STAPH- Combination of standard biochemical tests and fermentation tests which are the reference tests for the identification of staphylococci.

 6.      Rapid diagnostic tests -include Real-time PCR which is increasingly being employed in clinical laboratories.

 7.      Antibiotic Senistivity Tests (AST)

To be performed to help in deciding the treatment-which antibiotics to be used

 8.      Bacteriophage Typing

For epidemiological purpose

Phage typing -to trace the source of outbreaks of infections- to identify different strains of bacteria within a single species.

A culture of the strain is grown in the agar and dried. A grid is drawn on the base of the Petri dish to mark out different regions. Inoculation of each square of the grid is done by a different phage. The phage drops are allowed to dry and are incubated: The susceptible phage regions will show a circular clearing where the bacteria have been lysed, and this is used in differentiation

Phage type of the strain expressed by designations of all phages that lyse it

Eg., If a strain is lysed by phages a, b and c, it is designated phage type a/b/c

Treatment and Drugs

v  a) Antibiotic therapy – drug resistance common. Benzyl penicillin/methicillin/cloxacillins. For resistant strains, Vancomycins or teicoplanins.

For superficial mild lesions- topical applications of bacitracin/antispetics like chlorhexidine/mupirocin, sufficient.

In resistant cases, rifampicin along with other oral antibiotic found effective

v  b) Wound drainage

v  c)  Device removal (catheters etc)

v  d) Removal of dead tissue

Prevention

v  a) Wash your hands

v  b) Keep wounds covered

v c) Reduce tampon risks

v  d) Avoid sharing personal care items

v  e) Cooking and storing food properly

 

MRSA • Most strains of S.aureus, even those acquired in community, are penicillin resistant – Resistance is attributable to beta-lactamase production due to genes located on extrachromosomal plasmids. • Some are resistant to the newer beta-lactamase resistant semisynthetic penicillins, such as methicillin, oxacillin, nafcillin. – Resistance is due to presence of unusual penicillin-binding protein(PBP)in the cellwall of resistant strains • Infection with MRSA is likely to be more severe and require longer hospitalization, with incumbent increased costs than infection with a methicillin susceptible strain.

CONS • Coagulase Negative Staphylococci(CONS) that are commonly implicated as pathogens include • Staphylococcus epidermidis: causes infection of native heart valves and intravascular protheses. • Staphylococcus saprophyticus: causes urinary tract infections, mainly in sexually active women. • CONS that are less commonly implicated as pathogens include: S.hominis, S.haemolyticus, S.cohnii, s.lugdunensis, S.saccharolyticus, S.schleiferi, S.simulans and S. warneri