Detection of Methicillin-Resistant Staphylococcus aureus Using a New Medium, BBL™ CHROMagar™ MRSA, Compared to Current Methods

S. KIRCHER, N. DICK, V. RITTER, K. STURM AND P. WARNS BD Diagnostics • 7 Loveton Circle • Sparks, MD, USA 21152 REVISED ABSTRACT BBL™ CHROMagar™ MRSA (CMRSA) is a unique medium designed for the direct detection of MRSA from specimens. CMRSA incorporates cefoxitin and chromogenic substrates as indicators of methicillin resistant S. aureus. As S. aureus grow, they convert the specific chromogenic substrate to a colored product. CMRSA was compared to BBL™ Oxacillin Screen Agar (Ox Screen), disk diffusion using the 1 μg Oxacillin (OX) and 30 μg Cefoxitin (FOX) disks, and Penicillin Binding Protein 2’ Latex Agglutination Test (PBP2’) (Oxoid Limited, Hampshire) for the identification of mecA- positive S. aureus (MRSA). A total of 413 isolates were evaluated: 203 mecA-positive S. aureus; 93 mecA-negative S. aureus (MSSA); 86 coagulase-negative staphylococci (CNS); and 31 other organisms. All MRSA were inoculated onto the CMRSA plate at 10³CFU/mL; MSSA, CNS and other species were inoculated at 10⁵ CFU/mL. CMRSA plates were incubated at 35-37°C and read at 24 h; if negative, plates were reincubated an additional 24 h. Ox Screen and OX/FOX disk diffusion were prepared using a 0.5 McFarland suspension of MRSA and MSSA. The Ox Screen was spot-inoculated with a 10 μL drop and incubated at 33-35°C for a full 24 h. OX and FOX disk diffusion were inoculated per NCCLS-recommended procedures (M2), incubated at 33-35°C and read at 24 h. PBP2’ latex was performed per the manufacturer’s instructions. At 24 h, CMRSA produced a sensitivity of 82% and a specificity of 99.5%. At 48 h, the sensitivity increased to 91% and the specificity remained relatively constant at 98%. The sensitivity of the Ox Screen, OX disk diffusion, FOX disk diffusion and PBP2’ was 96%, 98%, 99.5% and 99.5%, respectively. The specificity for each procedure was 83%, 83%, 100% and 98%, respectively. CMRSA provides reliable identification of MRSA, and in addition, may offer a direct screen for clinical and surveillance samples. INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA) are a major cause of nosocomial infections. Selection of these organisms has been greatest in the healthcare environment; however, MRSA have also become more prevalent in the community setting.

Control of transmission from patient to patient or healthcare worker to patient is critical. It has been demonstrated that an active surveillance program, with steps taken to eliminate carriage and strict contact precautions can dramatically reduce infection rates.

BBL™ CHROMagar™ MRSA (CMRSA) is a selective and differential medium designed for the direct detection of MRSA from sources, such as, anterior nares and throats. CMRSA permits detection and identification of MRSA through the incorporation of specific chromogenic substrates and cefoxitin. MRSA strains will grow in the presence of cefoxitin and produce mauve-colored colonies resulting from hydrolysis of the chromogenic substrate. A proprietary blend of chromogens offers differentiation of methicillin-resistant S. aureus from other organisms. Additional selective agents are incorporated for the suppression of gramnegative organisms, yeast and some gram-positive cocci.

BBL™ CMRSA, Oxacillin Screen Agar (Ox Screen), oxacillin (OX) and cefoxitin (FOX) disk diffusion and PBP2’ Latex were evaluated for the detection and differentiation of MRSA from non-MRSA strains. MATERIALS AND METHODS Materials

• BBL™ CHROMagar™ MRSA (BBL, Cat. No., Available October 2004)
• Oxacillin Screen Agar (BBL, Cat. No. 221952)
• Mueller Hinton II Agar (BBL, Cat. No. 221275)
• Oxacillin 1 μg antibiotic disk (BBL, Cat. No. 231319)
• Cefoxitin 30 μg antibiotic disk (BBL, Cat. No. 231591)
• Trypticase™ Soy Agar with 5% Sheep Blood (TSA II) (BBL, Cat. No. 221261)
• Penicillin Binding Protein 2’ Latex Agglutination Test (PBP2’) (Oxoid Limited, Hampshire)

Methods CMRSA: MRSA strains were inoculated at a concentration of 10³ CFU/mL; all other organisms were inoculated at a concentration of 105 CFU/mL. Media were incubated aerobically at 35–37°C and examined at 24 h; if negative, reincubated an additional 24 h per the product specification. TSAII (sheep blood agar) was also included as a growth control. Colonies of MRSA appear as mauve; other organisms, if not inhibited, appear colorless, white, blue or blue/green.

OX SCREEN: S. aureus strains were inoculated onto the Oxacillin Screen Agar using a 10 μL drop of a 0.5 a McFarland Suspension, per the package insert instructions. Plates were incubated a full 24 h at 33–35°C. Isolates were scored as positive / MRSA (> 1 colony) or negative / not MRSA (no growth).

OX & FOX DISK DIFFUSION: 0.5 McFarland suspensions of S. aureus strains were inoculated to Mueller Hinton II Agar per NCCLS M2-A8. Following application of the antibiotic disks, plates were incubated at 33-35°C for a full 24 h. Zones of inhibition were measured to the nearest whole millimeter.

PBP2’ LATEX AGGLUTINATION: S. aureus strains were processed per the manufacturer’s instructions. Isolates were scored as positive / MRSA (agglutination of the test well), negative / not MRSA (no agglutination in the test well) or invalid, (agglutination in the control well).

MRSA	MSSA	Coagulase-negative Staphylococcus	Coagulase-negative Staphylococcus

RESULTS CMRSA A total of 413 organisms were evaluated on CMRSA. Of the 203 MRSA strains, CMRSA detected 166 following overnight incubation, for a sensitivity of 82%. The sensitivity increased, after an additional 24 h incubation, detecting 184, for a sensitivity of 91%. The specificity at 24 and 48 h was 99.5% (209/210) and 98% (206/210), respectively.

Of the 19 false negative strains, 1 had an MIC to oxacillin of 2 μg/mL and the remaining 18 appeared to be sensitive to selective agents in the medium base. The base-sensitive strains were obtained from a single site and may represent a rare finding. The 4 false positives included a S. haemolyticus, 2 MSSA (one with an oxacillin MIC ≥ 4 μg/mL) and one C. meningosepticum.

OXACILLIN SCREEN AGAR This medium is designed for the detection of MRSA using known S. aureus strains; therefore, only S. aureus were evaluated. The resulting sensitivity and specificity were 96% (195/203) and 83% (77/93), respectively. The low specificity was primarily due to difficulty interpreting growth versus inoculum for many of the MSSA strains. Of the 16 false positive strains, 5 had an oxacillin MIC ≥ 4 μg/mL. The remaining 11 false positive strains had MICs ranging from < 0.12 to 2 μg/mL. OXACILLIN DISK DIFFUSION Only S. aureus strains were evaluated by this method. The following interpretative ranges were used: R ≤ 10 mm; I 11–12 mm; S ≥ 13 mm, per NCCLS M100-S14. The OX disk detected 199/203 MRSA strains for a sensitivity of 98%. One intermediate was observed with an MRSA strain. This isolate had an OX MIC = 1 μg/mL. The specificity of this method was 83% (77/93). Seven of the 16 false positive organisms were the same as those producing false positives on the Ox Screen Agar. Twelve of the false positive organisms had oxacillin MICs ranging from < 0.12 to 2 μg/mL. Four had MICs ≥ 4 μg/mL. CEFOXITIN DISK DIFFUSION Only S. aureus strains were evaluated. The new interpretative ranges, per NCCLS M100-S14, were used to distinguish MRSA from MSSA. S. aureus with a zone diameter of ≤ 19 mm were scored as resistant and those with a zone diameter of ≥ 20 mm were reported as susceptible. The resulting sensitivity and specificity were 99.5% (202/203) and 100% (93/93), respectively. One MRSA strain produced a zone diameter of 24 mm. This isolate also had an MIC to oxacillin of 2 μg/mL.

PBP2’ LATEX Only S. aureus strains were tested with the PBP2’ Latex kit. PBP2’ was performed per the package insert instructions. The resulting sensitivity and specificity were 99.5% and 98%, respectively.

Coagulase-negative staphylococci

MRSA / MSSA Strains

CONCLUSIONS

• In 24 h, CMRSA had a positive predictive value (PPV) of 99% and a negative predictive value (NPV) of 85%.
• The oxacillin-based tests had a lower specificity due to their inability to distinguish mecA-positive from mecAnegative S. aureus strains.
• Cefoxitin disk diffusion and PBP2’ provide a reliable means of identifying MRSA; however, these require prior isolation and identification.
• CMRSA is the only method with the potential to recover and identify MRSA in a single step thereby reducing the time to detection.