Comparative study on detection of MRSA using oxacillin agar screening method, cefoxitin disc diffusion method and mecAgene by PCR among clinical isolates of Staphylococcus aureus.

 

F. Mariyam Niyas¹, Dr. Gopinath P²

¹BDS 2nd year, Saveetha Dental College, Saveetha University, Chennai.

²Senior Lecturer, Department of Microbiology, Saveetha Dental College, Saveetha University, Chennai.

 

ABSTRACT:

Cefoxitin is a good marker of the mecA regulatory system. It is being recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) using disk diffusion testing. The aim of our study was to evaluate the efficacy of cefoxitin disc diffusion test to characterize MRSA and compare it with oxacillin agar screening and detection of mecA gene by PCR. We have observed 100% sensitivity and specificity in cefoxitin disc diffusion method over detecting mecA gene by PCR. Results of cefoxitin disc diffusion test is in concordance with the PCR for mecA gene. Thus, the test can be an alternative to PCR for detection of MRSA in resource constraint settings.

 

 

INTRODUCTION:

Staphylococcus aureusis one of the most frequent bacterial pathogen in humans. It causes skin infections, osteoarthritis and respiratory tract infections in the community, as well as postoperative and catheter-related infections in hospitals^[1]. Methicillin-Resistant Staphylococcus aureus (MRSA) is an important bacterial pathogen causing nosocomial and community onset infections ^[2-3]. The prevalence of MSRA has increased in many parts of the world causing serious infections in hospitals that pose a serious burden in terms of medical and socio-economic costs and cause significant morbidity and mortality ^[4].

The rising colonization rates of S. aureus lead to the increasing of infection rates in the community and in hospitals. The consequences to the health care system are longer hospital stays and greater costs, which approximately double the expenditure per patient ^[5]. The patient risks include significantly higher mortality and morbidity rates with invasive MRSA infection. Health care workers may carry MRSA on their hands or clothes following their contact either with to asymptomatic carriers or patients who have clinical infection^[6].

 

Resistance of Staphylcocci to methicillin and all the beta-lactam antibiotics is associated with the low affinity of a penicillin binding protein PBP2a, which is not present in susceptible staphylococci ^[7-8]. This protein is encoded by the mecA gene, which is located in the mec region and is DNA of foreign origin ^[9].

 

MATERIALS AND METHODS:

Bacterial isolates:

A total of 20 clinical isolates of S.aureus were collected from different clinical specimens of patients attending Saveetha Medical Collage and Hospital. They were processed for a battery of standard biochemical tests and confirmed. Isolates were preserved in semisolid trypticase soy medium and stored at 4ºCuntil further use.

 

Antibiotic susceptibility test:

Antibiotic susceptibility testing was determined for these isolates to the following antibiotics such as penicillin, erythromycin, clindamycin, ciprofloxacin, tetracycline, cotrimoxazole and linezolid. These antibiotics were procured from Himedia, Mumbai. This was performed by Kirby-bauer disc diffusion method as per CLSI guidelines ^[10].

 

Detection of MRSA

Oxacillin agar screening method

Mueller-Hinton agar (MHA) plates containing 4% NaCl and 6 µg/ml of oxacillin were prepared. Plates were inoculated with 10 µL of 0.5 McFarland suspension of the isolate by streaking in one quadrant and incubated at 35 ⁰ C for 24 h. Plates were observed carefully in transmitted light for any growth. Any growth after 24 h was considered oxacillin resistant ^[11].

 

Cefoxitin disc diffusion method

All the isolates were subjected to cefoxitin disc diffusion test using a 30µg disc. A 0.5 McFarland standard suspension of the isolate was made and lawn culture done on MHA plate. Plates were incubated at 37 ⁰ C for 18 h and zone diameters were measured. An inhibition zone diameter of ≤ 19 mm was reported as oxacillin resistant and ≥20 mm was considered as oxacillin sensitive ^[12].

 

Detection of mecA gene by PCR

S. aureus isolates were detected for the presence of mecAgene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 1. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. A 50 µl PCR reaction consisted of 45 µl of master mix containing PCR buffer (1X), dNTP mix (0.2 mM of each), primer (0.5 µM), Taq DNA polymerase (0.25 U), and MgCl2 (1.5 mM) with 5 µL of template DNA. Cycling conditions were started at 94°C for 3 minutes followed by 30 cycles of denaturation at 94°C for 55 seconds, annealing at 50°C for 45 seconds, and extension at 72°C for 1 minute and final extension step at 72°C for 2 minutes. PCR products were visualized on 1.5% agarose gel with ethidium bromide dye under UV trans illuminator. A 100bp ladder was including in all the gel analysis.^[13]

 

 

Table 1: Gene sequence of mecA gene

 

 

RESULTS:

Sample wise distribution of clinical isolates of S. aureus

Of 20 clinical isolates of S. aureus, 8/20 (40%) were obtained from pus, 6/20 (30%) were from wound, 4/20 (20%) and 2/20 (10%) were from blood and sputum respectively (Figure 1).

 

Figure 1: pie chart showing the sample wise distribution of S. aureus

 

Antibiotic susceptibility pattern

We have observed a varied pattern of sensitivity among one S. aureus isolates. There was complete resistance observed for penicillin (100%), 9/20(45%) isolates were shown to the resistant to erythromycin, 6/20(30%) were to cotrimoxazole, 4/20(20%) were to linezolid followed by 3/20(15%) were resistant to ciprofloxacin and clindamycin respectively (Table 1) (Figure 2).

 

 

Table 2: Results of antibiotic susceptibility pattern of S.aureus

Antibiotics	Sensitive (%)	Intermediate (%)	Resistant (%)
Penicillin	0	0	20(100)
Erythromycin	14(70)	4(20)	2(10)
Clindamycin	15(75)	2(10)	3(15)
Ciprofloxacin	9(45)	8(40)	3(15)
Tetracyclin	14(70)	4(20)	2(10)
Cotrimoxazole	10(50)	4(20)	6(30)
Linezolid	10(50)	6(30)	4(20)

 

Figure 2: Representative picture showing antibiotic sensitivity pattern of S.aureus

 

 

 

Figure 3: Representative picture showing MRSA by Cefoxitin disc diffusion method

MRSA detection by cefoxitin disc diffusion and oxacillin agar screening methods:

Using cefoxitin distribution method 15/20(75%) were found to be MRSA isolates. Whereas, oxacillin agar screening method yielded 2/20(10%) of MRSA isolates (Figure 3 and 4).

Result of mecA gene in Staphylococcus aureus by PCR:

Out of 20 clinical isolates of S. aureus, 15/20 (75%) of isolates were found to be positive for mecA gene.

 

Figure 4: Representative picture showing MRSA by Oxacillin agar screening method

	L1          L2           L3           L4          L5

 

 

 

L2 – 100bp ladder, L4 – mecA gene positive

 

Figure 5: Representative gel picture showing mecA gene positive

 

Table 3: Results of MRSA detection using 3 different methods

S.aureus isolates	Cefoxitin disc diffusion method	Oxacillin agar screening method	mecA by PCR
20(100%)	15(75%)	2(10%)	15(75%)

 

 

DISCUSSION:

Detection of MRSA is important for patient care and appropriate utilization of infection control resources. Methicillin-resistant S. aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains ^[11].

 

Detection of mecA gene or its product, penicillin binding proteins (PBP2a), is considered to be the gold standard for MRSA detection ^[14]. Recent studies documented that disc diffusion testing using cefoxitin disc is more superior to most of the currently recommended phenotypic methods like oxacillin disc diffusion and oxacillin screen agar testing and is now an accepted method for the detection of MRSA by many reference groups including CLSI ^[15]. The prompt and early detection of methicillin resistance is very important in the prognosis of infections caused by S. aureus. In thisstudy, we attempted to evaluate different methods for detecting MRSA.

 

During the last many years, the CLSI has attempted to improve the accuracy of detecting mecA strains among S. aureus. In our study, the observations using the CLSI disc diffusion criteria to define resistance (cefoxitin zone diameters of ≤19 mm for resistance and ≥20 mm for sensitivity), the sensitivity and specificity were 100% in the 20 strains tested in the study whereas the results of oxacillin screen agar were not so accurate. Results of cefoxitin disc diffusion test is in concordance with the PCR for mecA gene, and thus the cefoxitin disk diffusion method is very suitable for detection of MRSA and the test can be an alternative to PCR for detection of MRSA in resource constraint settings.

 

CONCLUSION:

Although the number of isolates used in this study are less, this study provides an evidence that cefoxitin can be used as an accurate marker in routine susceptibility testing. In addition, the results have shown 100% sensitivity and specificity as compared to mecA gene detection by PCR. Hence, it can be used as an alternative to the technically demanding PCR.

 

 

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Accepted on 15.07.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(9):1317-1320.