J Med Microbiol 56 (2007), 500-503; DOI: 10.1099/jmm.0.46981-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Comparison of four chromogenic media for culture-based screening of meticillin-resistant Staphylococcus aureus
Abdessalam Cherkaoui1,
Gesuele Renzi1,
Patrice François2 and
Jacques Schrenzel1,2
1 Clinical Microbiology Laboratory, University of Geneva Hospitals (HUG), Service of Infectious Diseases, CH-1211 Geneva 14, Switzerland
2 Genomic Research Laboratory, University of Geneva Hospitals (HUG), Service of Infectious Diseases, CH-1211 Geneva 14, Switzerland
Correspondence
Patrice François
patrice.francois{at}genomic.ch
Received 3 October 2006
Accepted 14 December 2006
Meticillin-resistant Staphylococcus aureus (MRSA) are responsible for nosocomial and community-acquired infections. Detection of MRSA is of utmost importance for the adaptation of infection control and therapeutic strategies. To date, selective agar plates constitute the standard routine method for reliable detection of this worldwide infectious problem. The performance of four different chromogenic media was evaluated in this study for MRSA detection and identification on >240 consecutive swab samples. The results indicate that primary plating on MRSASelect or MRSA ID is more sensitive than screening with oxacillin-based culture media. In addition, the utilization of cefoxitin- or cephamycin-containing plates reduces significantly the number of required confirmatory tests. Several selective agar plates allowing the identification of S. aureus are commercially available. However, their respective performances under real conditions of utilization are heterogeneous, underlining the absence of a gold standard medium for MRSA screening.
Abbreviations: MRSA, meticillin-resistant Staphylococcus aureus.
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INTRODUCTION
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Staphylococcus aureus is one of the most commonly identified pathogens in human medicine and is a major cause of nosocomial infections (Boyce et al., 1983) and community-acquired infections (Naimi et al., 2003; Said-Salim et al., 2003). Resistance to meticillin, reported for the first time in 1961 (Barber, 1961), is now widespread in hospitals all over the world (Doebbeling, 1995), and recently decreased susceptibility to vancomycin was reported (Kuroda et al., 2001; Samra et al., 2005). The rapid and reliable identification of meticillin-resistant S. aureus (MRSA) appears nowadays to be essential for appropriate patient care, control of strain spreading and use of adapted empirical antimicrobial guidelines. Active surveillance for MRSA revealed an efficient and recommended strategy to control nosocomial MRSA infections (Arnold et al., 2002; Boyce, 2001), but requires robust and rapid identification. To date, several molecular assays have been developed and evaluated (Cuny & Witte, 2005; Francois et al., 2003; Huletsky et al., 2004; Warren et al., 2004). Despite recent commercialization of some molecular methods, the standard procedure used in routine laboratories for reliable MRSA identification remains based on cultures using selective agar media. Numerous reports in the literature have described screening media for MRSA identification showing variable performance. Phenotypic methods based on oxacillin-containing medium are not satisfactory in terms of sensitivity and specificity, and are sensitive to incubation temperature (Apfalter et al., 2002; Skov et al., 2006), time (Blanc et al., 2003; Simor et al., 2001) and inoculum density (Smyth et al., 2001). Therefore, MRSA identification using cefoxitin- or cephamycin-containing media appears more robust (Felten et al., 2002; Skov et al., 2003) and is now recommended by the CLSI (Clinical and Laboratory Standards Institute, 2005).
The purpose of our study was to compare the performance of four chromogenic media for the screening of MRSA. We evaluated oxacillin-resistance screening agar base (ORSAB; Oxoid); MRSA ID (bioMérieux); Chromogen oxacillin S. aureus (Axon Lab) and MRSASelect (Bio-Rad) on 247 consecutive swab samples collected in a tertiary university hospital. Our results show that the performances of these media vary in terms of sensitivity, and often warrant further confirmatory testing.
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METHODS
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Study population and specimens.
Two hundred and forty-seven consecutive patients were screened for the presence of MRSA. For each patient, nasal and inguinal swabs were obtained and subjected to analysis (Copan Italia).
Culture media.
MRSASelect (Bio-Rad) is composed of a selective base medium containing a cephamycin derivative (proprietary formulation) and characterizes S. aureus colonies by a specific mauve colour. MRSA ID (bioMérieux) similarly combines chromogenic identification, based on the spontaneous green colouration produced by a specific enzyme of S. aureus, with the use of 4 mg cefoxitin l–1. ORSAB (Oxoid) uses aniline blue to detect mannitol fermentation, resulting in intense blue colonies of S. aureus. The formula of the agar base is: 11.8 g peptone l–1, 9.0 g yeast extract l–1, 10.0 g mannitol l–1, 55.0 g NaCl l–1, 5.0 g LiCl l–1, 0.2 g aniline blue l–1 and 12.5 g agar l–1. This agar base medium is supplemented with selective compounds to detect oxacillin resistance: 50 000 IU polymyxin B l–1 and 2 mg oxacillin l–1. Chromogen oxacillin S. aureus (Axon-Lab) is composed of a base medium characterizing S. aureus colonies by a specific pink-mauve colour. Colistin-Salt (CS) broth consists of a brain heart infusion broth supplemented with 10 mg colistin l–1 and 25 g NaCl l–1. MHO broth consists of Mueller–Hinton broth supplemented with 6 mg oxacillin l–1 and 45 g NaCl l–1.
Bacteriological procedures.
Particular attention was paid to the design of this study. Each of the four technologists had to evaluate every tested media, to prevent user-related bias. To inoculate equivalent sample volumes on the different media, swabs were extracted with dedicated Eppendorf tubes (Swab Extraction Tube System; Roche), following the manufacturer’s instructions. For each patient, collected fluids from the two swabs were pooled and supplemented with sterile NaCl (0.9 %) to a final volume of 250 µl. An identical volume of 50 µl was used to inoculate each of the four selective media. The remaining 50 µl were inoculated into CS broth as a final backup evaluation procedure. All cultures were incubated aerobically at 35 °C. For all samples showing negative results at 24 h on the four media, CS broth was used to re-inoculate another set of selective media, in order to evaluate the utility of an enrichment step before plating onto selective agar.
Identification of MRSA.
Mauve colonies on MRSASelect, green colonies on MRSA ID, pink-mauve colonies on Chromogen oxacillin S. aureus and blue colonies on ORSAB were considered as presumptive MRSA isolates and subcultured onto Columbia blood agar. Any colony of the expected colour was considered as a positive result, to benefit from optimal assay sensitivity. Confirmation of MRSA isolates was achieved by a combination of tests, including growth in MHO broth, Staphychrom II (International Microbio) (Fonsale et al., 2004) and a duplex quantitative PCR to evaluate the presence in similar abundance of the mecA gene and that of the femA gene specific for S. aureus (Francois et al., 2003).
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RESULTS AND DISCUSSION
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A total of 247 consecutive samples submitted to the bacteriology laboratory were screened for the presence of MRSA, each sample consisting of two separate swabs (nostril and groin). All samples were obtained from adults admitted to our institution: 50 % of samples originated from patients hospitalized in long-term healthcare facilities, 40 % from various medical wards, whereas the remaining 10 % were referred by the surgical intensive care unit. Pooled liquid obtained by swab centrifugation was equally inoculated on the four selective media and in a CS enrichment broth. From the 247 specimens tested in this study, a total of 70 MRSA strains were isolated by the combination of all the culture-based media (Table 1
). MRSA prevalence was 28 % in our collection, encompassing samples collected during admission screening, reflex testing of neighbouring patients of MRSA carriers and decontamination controls. After 24 h of incubation, CS enrichment broth was subcultured onto the four media. Relative sensitivity was calculated based on positive cultures observed on any of the four media, directly, or including the use of CS broth enrichment (Table 1). When solid culture media were directly inoculated, relative sensitivities at 24 h were 76, 76, 1 and 77 %, and reached 87, 90, 53 and 91 % at 48 h, for ORSAB, MRSA ID, Chromogen oxacillin S. aureus and MRSASelect, respectively. When combined with the CS enrichment broth, the sensitivities achieved were 91, 94, 53 and 96 %, respectively. Thus, CS enrichment broth marginally contributed to improve screening sensitivity. The additional reporting delay imposed by its use revealed it was of questionable benefit, when considering the different media tested in this study.
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Table 1. Sensitivity and specificity of chromogenic media for detecting MRSA isolates among 247 clinical specimens
Overall, 70 MRSA isolates were identified in our population, equating to 28 % prevalence.
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To evaluate the discrimination provided by each medium and quantify its impact on the workload, we determined the number of subcultures required to assess MRSA identification. Subcultures refer to the process of strain isolation and confirmation testing for any colony morphology of the expected colour that grows on selective medium. Specificity was determined according to the results of these confirmatory tests.
Two hundred and forty-one pink-mauve (positive) colonies that grew on Chromogen oxacillin S. aureus were subsequently identified as meticillin-resistant coagulase-negative staphylococci, reflecting insufficient concentrations of the selecting agent, oxacillin. The poor specificity of this medium precludes its use for the rapid detection of MRSA. ORSAB performed better than Chromogen oxacillin S. aureus, but was revealed as slightly less sensitive and much less specific than MRSA ID and MRSASelect. Values obtained in this study are in accordance with another evaluation (Apfalter et al., 2002). ORSAB medium presents limitations for surveillance applications (Becker et al., 2002) not only due to lower sensitivity, but also because some coagulase-negative staphylococci (mainly Staphylococcus haemolyticus, a frequent skin colonizer) appear blue (Becker et al., 2002). Thus, utilization of this plate warrants confirmatory tests for MRSA identification and should rather be considered in high prevalence settings (Becker et al., 2002; Simor et al., 2001). Sensitivities of MRSA ID and MRSASelect were comparable and outperformed that of ORSAB, likely due to the substitution of oxacillin by a cephamycin (Perry et al., 2004). The few MRSA strains that failed to be detected by MRSASelect and MRSA ID can be explained by shortcomings of selective media when inocula are very low. The good discrimination of green colonies on MRSA ID and mauve colonies on MRSASelect facilitated the early recognition of potential MRSA isolates. However, the evaluation of cost–benefit performances should also integrate the number of required subcultures. The number of subcultures at 24 and 48 h reached 118 and 51, 71 and 22, 170 and 71, and 66 and 51, for ORSAB, MRSA ID, Chromogen oxacillin S. aureus and MRSASelect, respectively. Interestingly, MRSA ID grew larger colonies at 24 h and maintained specificity >95 % after 48 h incubation, thus providing the best balance in terms of sensitivity and costs in our hands.
This study has several limitations. First, collecting fluids from the two swabs and supplementing them with sterile saline provides fair comparison of the media, but slightly differs from routine protocols when swabs are directly streaked on agar. Second, such results should be formally compared to other chromogenic assays, including ChromAgar/MRSA (ChromaAgar). This medium, where MRSA colonies appear mauve while other bacteria display different colours, has been extensively tested and shows appreciable sensitivity and specificity (Diederen et al., 2005; Hedin & Fang, 2005; Kluytmans et al., 2002), fulfilling routine laboratory needs in terms of rapidity and sensitivity (Flayhart et al., 2005). Third, the selectivity of each chromogenic medium is based on a distinct and different biochemical feature of S. aureus. Thus, the performance of each medium depends also on the possession and expression of the respective enzyme that is assessed by the chromogenic assay.
Taken together, our results indicate that primary plating on MRSASelect or MRSA ID is convenient and time-saving for the presumptive identification of MRSA in routine microbiology. Interpretation of colony colours is easy and false-positive detection is rare when plates are incubated for 48 h. Combined use of MRSASelect or MRSA ID with a confirmatory MRSA identification assay (e.g. Staphychrom II or mecA determination) appears optimal in terms of results. Such a strategy is now routinely implemented in our institution, in combination with our rapid molecular screening assay (Francois et al., 2003), which is employed whenever carriage status has to be known within a few hours. In the present study, this rapid molecular assay allowed us to rapidly identify for confirmatory purpose the totality of MRSA isolates.
Conclusion
Despite similar principles of identification and equivalent reagent costs, selective agar-based media revealed great differences in terms of performance, affecting both specificity and sensitivity. Among the four commercial products tested in our study, MRSASelect and MRSA ID appeared superior but still require confirmatory testing to assess the identification of MRSA in clinical samples.
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ACKNOWLEDGEMENTS
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This work was supported by grants from the Swiss National Science Foundation nos. PP 00B-103002/1, 632-057950.99 (J. S.) and 404940-106296/1 (P. F.), and from the University of Geneva Hospitals (CI 70889, CI 70903 and CI 70897).
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TOP
INTRODUCTION
METHODS
