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Dear Editor,
Methicillin resistant Staphylococcus aureus
(MRSA) is an important nosocomial pathogen.
1
Since
most of these strains are multidrug resistant, management
of infections is difficult. Rapid and accurate detection
of MRSA is important for proper management and
prevention of transmission. Heterogeneous nature of
methicillin resistance is a limitation to accurate detection
of MRSA by routine testing methods.
1
We report here
a simple broth-disk technique for the detection of
methicillin resistance in S. aureus. This method is a
modification of a similar procedure described for
anaerobic bacteria.
2
Eighty strains of S. aureus isolated from clinical
samples in the Department of microbiology, Kasturba
Medical College, Mangalore, were studied for methicillin
resistance with disk diffusion,
3
agar screen,
4
agar
dilution
5
and broth-disk methods.
2
Disks of diameter 6.1 mm were prepared using
Whatman no. 1 filter paper and sterilized in hot air oven
at 140C for 1 hour. Oxacillin was diluted in distilled
water to get solutions of concentration 400 and 200 g/
mL. Antibiotic solution (0.01 mL per disk) was added to
disks to prepare disks of potency 4 and 2 g oxacillin.
The disks were stored at 4C.
For each test, two tubes each containing 1mL
Mueller Hinton broth with 2% NaCl was taken. One disk
each of potency 4 g and 2 g was added separately to
each tube and shaken well. To each tube, 0.01 mL of 1
in 200 diluted overnight broth culture of the test organism
was added. An inoculated broth containing no antibiotic
was used as growth control and a tube of uninoculated
broth was included as sterility control. All the tubes
were incubated at 35C for 24 hours and observed for
growth. S.aureus that could grow in broth containing 2
and 4 g/mL oxacillin was considered methicillin
resistant. S.aureus ATCC 25923 and ATCC 29213 were
used as controls in disk diffusion and dilution methods
respectively.
Of 80 S. aureus isolates tested, 28 (35%) were scored
methicillin resistant by disk diffusion whereas 32 (40%)
were scored methicillin resistant by broth-disk method.
All the strains which were methicillin resistant in broth-
disk method were positive by agar screen and agar
dilution, indicating reliability of the broth-disk technique
(Table).
Table : Comparison of various methods for the detection
of methicillin resistant and methicillin sensitive S.
aureus (n=80)
Methods No. of MRSA No. of MSSA
Disk diffusion 28 52
Agar screen 32 48
Agar dilution 32 48
Broth-disk 32 48
MRSA methicillin resistant S.aureus
MSSA methicillin sensitive S.aureus
The concentrations of oxacillin used were selected
based on breakpoint level for S. aureus (> 4 g/mL-
resistant and < 2 g/mL susceptible).
5
The procedure
is simple and cost-effective. The potency of the disk
can be maintained for 2 months if stored at 4C.
Considering the above advantages, we feel that the
broth-disk method can be conveniently used in
laboratories to detect MRSA.
References
1. Koneman EW, Allen SD, Janda WM,
Schreckenberger PC, Winn WC. Antimicrobial
susceptibility testing, chapter 15. In: Color Atlas
and Textbook of Diagnostic Microbiology, 5t h
edition (Lippincott, Philadelphia) 1997: 785.
2. Matsen JM. Antimicrobial susceptibility test
laboratory testing in support of antimicrobial
therapy. In: Gradwohls clinical laboratory methods
and diagnosis, vol.2, 8th edition, Sonnenwirth AC,
Jarett L. Eds. (BI Publications, New Delhi) 1990:1937-
1970.
3. National Committee for Clinical Laboratory
Standards: Performance standards for Antimicrobial
Disk susceptibility Tests. Approved standards (M2-
A5) Villanova, Pa, 1993.
Simple Broth-Disk Technique for the Detection of Methicillin Resistance in
Staphylococcus aureus
Indian Journal of Medical Microbiology, (2004) 22 (1):69-70
Correspondence
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www.ijmm.org
*Corresponding author
Received : 06-05-2003
Accepted : 29-05-2003
susceptibility testing. Approved standards (M100-
59) Wayne, PA 1999.
V Nishi, BR Raman, C Yengkokpam, *GK Bhat
Department of Microbiology, Kasturba Medical
College, Mangalore 575 001, Karnataka, India.
4. Chamber HF. Methicillin resistance in Staphylococci:
Molecular and biochemical basis and implications.
Clin Microbiol Rev 1997;10:781-791.
5. National Committee for Clinical Laboratory
Standards. Performance standards for antimicrobial
70 Indian Journal of Medical Microbiology Vol.22, No.1
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