Iranian Journal of Medical Microbiology

2.1. Samples Collection

During an 11-month period (1 August 2019 to 30 June 2020), a total of 220 Staphylococcus aureus strains were isolated from different clinical specimens including urine, blood, wound and respiratory tract specimens of patients who were admitted at Milad hospital of Tehran. Milad hospital is a 1000-beds tertiary care hospital affiliated by Iranian social security. All isolates of S. aureus were identified by routine laboratory methods such as colony morphology, Gram staining, catalase, coagulase, DNase test and other biochemical reactions.

2.2. Preparation of CHROM agar^TM.  Medium

The CHROM agar Company provided CHROM agar^TM for MRSA identification (Paris, France). The product composed of this medium included agar (15g/l), peptone (40g/l), NaCl (25 g/l) and proprietary chromogenic mix (3g/l), totally 82.5g/l. The culture media was prepared as guideline that recommended by manufacture. Briefly 82g of CHROM agar^TM MRSA was dispersed in 1 L of purified water and stirred completely. The mixture was autoclaved at 110ºC for 5 minutes and then cooled in water bath (45-50ºC).
In step two, aseptically, CHROM agar MRSA supplement (ref. SU620) was rehydrated in 20 ml of sterile water and mixed slowly. 1 ml of CHROM agar supplement was add to prepared CHROM agar medium and dispensed in sterile plates. After solidification, all plates were stored up to one month under refrigeration (2-8°C) until use. Quality control was performed by using microorganisms as recommended by manufacture.

2.3. Inoculation of Strains on CHROM Agar

 For testing of S. aureus one fine 24-hour colony isolates processed by direct streaking on CHROM agar MRSA. Plates were incubated for 12-18 hour. Growth of intense colonies with mauve color considered as MRSA according to manufacture instruction.

2.4. Susceptibility Test

For detection of MRSA isolates, disk diffusion test using Cefoxitin (30µg) was used according to CLSI instructions. Bacterial suspension in 0.9% NaCl solution at a density equivalent to 0.5 McFarland standards was prepared with 4-5 fresh colonies of S. aureus on blood agar and inoculated to Mueller-Hinton agar plates. Cefoxitin disk (ROSCO. Co Denmark) aseptically placed on Mueller-Hinton agars. Plates were incubated at 35ºC for 24 hours. Inhibition zone diameters were measured in millimeter and interpreted according to instructions.

2.5. Detection of the mecA Gene by Polymerase Chain Reaction

The DNA extraction process was performed by the rapid method (11). An overnight culture on blood agar plates was prepared. One colony of each sample was re-suspended in 25 µl of sterile distilled water. The suspension was then placed in a heating block (100ºC) in water for 15 min. Ten-microliter volumes from this suspension was directly used as a template for PCR amplification.  The 162 bp fragment of the Methicillin-resistant gene (mecA) was amplified using the primers mecA-F (5'-TCCAGATTACAACTTCACCAGG-3') and the mecA-R (5'-CCACTTCATATCTTGTAACG-3') (6). An aliquot of 10 µl of extracted DNA was added to 40 µl of PCR mixture consisting of PCR buffer (1.5X), Taq DNA polymerase (0.1 U/μlit), dNTP mix (0.25mM of each), MgCl₂ (1.5mM), the primer (0.3 pmol) and with the following thermal cycling profile: denaturation step at 94°C for 5 min, followed by annealing at 58 °C for 50 sec, extension at 72 °C for 50 sec and the final extension step at 72 °C for 10 min. Under a UV Transilluminator, the PCR products were visualized on a 1.5% agarose gel with ethidium bromide dye. Amplicons of 162 bp were found to be compatible with the amplification of the mecA gene (Fig. 1).
Methicillin resistant S aureus (MRSA ATCC 43300) and methicillin sensitive S. aureus (MSSA ATCC 25293) were used as positive and negative controls, respectively. These strains were provided from Iranian reference health laboratory.

S. aureus was found in 220 of the 74572 surveillance specimens sent to Milad Hospital's microbiology lab. Out of 220 patients, 56.4% were male and 43.6% female. PCR testing revealed that 105 (47.72%) of the 220 S. aureus isolates were positive for the mecA gene. Figure 1 illustrates the electrophoresis of PCR product produced from S. aureus mecA genes on an agarose gel. The Cefoxitin disc diffusion method showed the highest sensitivity and specificity among phenotypic methods. Table 1 compares the sensitivity and specificity of various phenotypic approaches to the PCR method as a gold standard process for MRSA detection.
Various laboratory methods including phenotypic and genotypic have been introduced for detection of MRSA (5, 6, 12). Although the molecular methods are rapid and have a high sensitivity, but these methods are not achievable in many microbiology laboratories. In contrast phenotypic methods are time consuming but give a comparable sensitivity and lower price. There are different phenotypic methods for detection of MRSA, which are widely used in microbiology laboratories. The main phenotypic methods are based on disk diffusion methods such as using   oxacillin and Cefoxitin disks. Other methods such oxacillin screen agar, latex agglutination (Penicillin binding protein 2a) and chromogenic media are also available in some laboratories (13-15).
The oxacillin disc diffusion method is one of the oldest methods for detecting MRSA in clinical microbiology laboratories. Recently studies have shown a lower reliability of oxacillin disk for detection of MRSA and therefore Cefoxitin disk is substituted for oxacilln to detection of MRSA. Cefoxitin disk diffusion method is highly recommended by CLSI and this method is very reliable method with high sensitivities and specificity.
There are several culture-based media for isolation and identification of MRSA. With the advent of chromogenic media, diagnosis of MRSA has undergone a revaluation. Chromogenic medium has a number of advantages for the enumeration, detection, and identification of S. aureus. CHROMagar was the first medium which introduced for detection of MRSA. Using of CHROMagar^TM leads to significant reductions in detection time and workload, which it necessitated a wide-scale patient screening to determine its clinical utility (9). Many studies have been shown a higher level of sensitivity and specificity in comparison oxacillin containing media. Their results of CHROMagar and cefoxitin disk diffusion method are comparable. In this study both methods have 100% sensitivity and specificity (9, 10, 16, 17). In Brennan et al., study, that was performed in Ireland, the sensitivity of chromogenic agar ranging from 98% to 100% (10) which is similar to our study. In another study has been conducted in university of Manitoba by Manickam et al., reported a sensitivity of 98% and a specificity of 100% (17).  Because of the changing epidemiology of MRSA, rapid detection of MRSA from clinical specimen is very important issue for infection control and effective patent manage.

 
Table 1. Comparison of PCR and phenotypic methods for detection of MRSA.

Specificity %	Sensitivity %	No of MRSA	Method
100	100	105	PCR for detection of mecA gene
100	100	105	Cefoxitin Disk (30 µg)
100	100	105	CHROMagarTM MRSA

Figure 1. Detection of mecA gene by PCR method. MRSA strains isolated from clinical specimens showing in lane 2 and 3 while lane 4 contain 100bp ladder, lane 5 is having positive control (S. aureus ATCC 33591) and lane 6 is negative control (S. aureus ATCC 29213).

 
 

Oure study revealed a high sensitivity and specificity for identification of MRSA by CHROMagar which comparable with result of Cefoxitin disk diffusion method. Both methods have 100% sensitivity and specificity in comparison with PCR as a gold standard method for detection of mecA.

 

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Conflicts of Interest

The authors have no conflict of interest.