Culture Based Methods Dedicated to MRSA Identification

Liquid cultures have been instrumental to the nascent field of microbiology and are still used routinely in laboratories to recover minute amounts of bacteria or are employed as backup media. For example, Mueller-Hinton supplemented by oxacillin is still currently used to detect or confirm presence of MRSA from swabs sampled for surveillance programs. This medium appears in the guidelines for the prevention and control of antibiotic-resistant organism from the NCCLS recommendations (NCCLS,2002). However, solid media are now commonly used for organism isolation and identification, allowing MRSA identification in approximately 24 h. Agar plates provide numerous advantages, such as the possibility for microbiologists to detect the presence of relevant colony morphologies, isolate them by sub-plating, and assess their purity on isolation plates. Pure isolates are essential for further phenotypic testing, including speciation (when required), antimicrobial susceptibility testing, and typing. Agar plates developed for MRSA isolation or detection represent a particularly active field for microbiology companies. To date, numerous selective media containing (3 -lactam antibiotics and chromogenic substances are commercially available. The general principles are simple and consist in providing selective medium supplemented in (i) Gramnegative growth inhibitor (required for samples containing mixed flora), (ii) antibiotic (allowing the selection of methicillin-resistant organisms only), and (iii) a chromogenic substrate allowing the specific detection of growing Staphylococcus aureus colonies. ORSAB plates (Oxoid Ltd; Basingstoke, UK), a solid variant of the liquid mannitol-salt medium containing oxacillin and aniline blue, allows detection of mannitol-fermenting organisms as blue colonies, due to medium acidification. This medium appears adapted to high-risk population (Simor et al., 2001), but presents limitation for surveillance applications (Becker et al., 2002) as some coagulase-negative staphylococci (mainly S. haemolyticus, a frequent skin colonizer) also appear blue (Becker et al., 2002). Thus, the use of this plate requires additional tests for robust identification. To date, ChromAgar/MRSA (ChromaAgar, Paris, France) stands among the most popular agar plates dedicated to the detection of MRSA. This medium, where MRSA colonies appear mauve while other bacteria display different colors (Fig. 24.2), has been extensively tested and shows appreciable sensitivity and specificity (Kluytmans et al., 2002; Diederen et al., 2005). Other chromogenic agar media showing interesting performance are MRSA ID (bioMerieux, Marcy l'Etoile, France), containing cefoxitin and a chromogenic substrate of a-glucosidase. Recent evaluation of this medium has shown improved performance compared with ChromAgar/MRSA (Perry et al., 2004). Comparable results were reported with MRSA Select (Bio-Rad, Reinach, Switzerland) (www.rapidmicrobiology.com/PG/MRSA.php). All these media represent appreciable improvement of the current situation in the field of MRSA screening and identification, albeit these culture-based methods require at least 20-24 h to yield identification results. During this period of time, infection control measures cannot be optimally applied. And, in case of empirical treatment, options include usually glycopeptide prescription leading to important costs and suboptimal use of last-barrier drugs.

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