What is the standard procedure of determining MRSA in hospitals or diagnostic centers?
Hi Nikhil,
E-strip is not the only method and besides to my knowledge i dont think it is endorsed by CLSI or EUCAST. You can broadly classify detection methods into phenotypic and genotypic. In the latter you simply look for the presence or absence of the mecA gene associated withoxacillin resistance.
In the phenotypic method, you have two choices (here we use cefoxitin resistance as a surrogate marker for methicillin resistance or presence of the mecA gene).
1) Disk method: Use a 30 mcg oxacillin disk on Mueller Hinton agar which has been streaked with a 0.5 MacFarland opacity standard of the test organism. Incubate at 35 degrees celsius for 16-18 hours and measure the zone size. If less or equal to 21mm, it is considered resistant. If more than that, then sensitive. You must be very strict as to the quality of the media and the volume of medium in the plate i.e. 25ml approx. Also use positive and negative controls.
2) Broth micro-dilution: You need cation adjusted Mueller Hinton broth with 4 mcg/ml of cefoxitin. Incubate at the same temperatire as the disk method for 16-20 hours. Growth in tubes with more than and equal to 4 mcg/ml is positive for MRSA.
I hope this helps.
Michael.
The easiest way is to make a simple PCR for the mecA gene. We are doing this here in our practical student courses.
The definitive method is the detection of mecA gene by PCR. There are also fast methods available for detecting PBP2a directly. You also can use surrogate markers based on in vitro susceptibility tests to oxacillin or cefoxitin according CLSI or EUCAST guidelines.
In the clinical setting, the preliminar diagnosis is usually made through in vitro susceptibility tests to oxacillin and cefoxitin, and the can be confirmed by PCR methods.
Dear Nikhil,
The best way to differentiate is really by PCR as highlighted by Dr. Bellido. Please check this kit below:
http://www.bd.com/geneohm/english/products/pdfs/MRSA_ACP_Amplification_Kit_PI.pdf
Rubens
Thank you @Michael @ Dr. Bellido @Demircioglu & @Monte-Neto for your inputs.
Actually we have tried using only mecA gene detection and have found that the gene was amplified even MSSA isolates (MICs
What about Fatty acid profiling of MRSA & MSSA isolates. I have read a paper on that but am not able to get the full text of the same.
The paper is titled: Comparative study of the cellular fatty acids of methicillin-resistant and -susceptible Staphylococcus aureus by Asai S (APMIS. 1993 Oct;101(10):753-61.)
If anyone has a copy of the same could you share it with me.
Thank you Michael, will check the article that you have mentioned. Also do you know anything about the fatty acid profiling of MRSA. I am planning to start work on that but wanted a little more literature on it.
Nikhil,
Sorry could not answer earlier, but i have absolutely no knowledge of fatty acid profiling of bacteria other than mycobacteria (of which again i know a bare minimum). Sorry i cant help.
Regards,
Michael.
Staphylococcus aureus infections can be resistant to methicillin - an antibiotic - or susceptible to it. MRSA is resistant to methicillin while MSSA is susceptible. From thisreasons E-test or other antibiotic susceptibility can be used for diffentiation of MRSA and MSSA
L. Langsadl, Dept.hygiene a hospital epidemiology, University Hospital Bratislava,
Slovak republic
pcr method to detect gene encoding methicillin resistant it's simple and gold standard method, you can use conventional, multiplex or real time PCR to detect these gene. many journal articles discuss detection of MRSA by PCR
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