Detection of methicillin-resistance gene in Staphylococcus epidermidis strains isolated from patients in Al-Zahra Hospital using polymerase chain reaction and minimum inhibitory concentration methods
Ebtehaj Pishva1, Seyed Asghar Havaei1, Firouz Arsalani1, Tahmineh Narimani1, Amir Azimian1, Mojtaba Akbari2
1 Department of Microbiology, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran
2 Vice chancellor for research, School of medicine, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Submission||07-Aug-2012|
|Date of Acceptance||26-Aug-2012|
|Date of Web Publication||06-Mar-2013|
Seyed Asghar Havaei
Department of Microbiology, Medical School, Isfahan University of Medical Sciences, Isfahan
Source of Support: This article is obtained from a MS thesis in Microbiology
which was conducted in Isfahan University of Medical Sciences., Conflict of Interest: None
Background: In recent years, antibiotic resistance of Staphylococcus epidermidis to methicillin has significantly increased, making it essential to study resistance to methicillin, which is a determining factor in the appropriate treatment pattern. The purpose of this study was to identify methicillin-resistant genes in S. epidermidis strains using polymerase chain reaction (PCR) and to determine their mean minimum inhibitory concentration (MIC) to methicillin using E-test method.
Materials and Methods: MIC was determined on 146 samples of S. epidermidis using E-test method. Moreover, all samples were tested for the presence of mecA gene using PCR.
Results: PCR test showed 75.34% of the samples to contain mecA gene. Methicillin resistance test was performed using E-test on all the samples, which showed resistance in different dilutions.
Conclusion: The frequency of mecA gene in S. epidermidis isolates was 75.34%. Among the various applied tests used for determining methicillin resistance, sensitivity and specificity of PCR were the highest and reached 100%. Sensitivity and specificity were found to be 95.3% and 94.7%, respectively, for phenotypic test (E-test) and 86.5% and 80.9%, respectively, for disk diffusion method. Based on the above results, it seems that resistance of S. epidermidis to methicillin is on the rise, and therefore more research is warranted.
Keywords: E-test, mecA gene, methicillin, Staphylococcus epidermidis
|How to cite this article:|
Pishva E, Havaei SA, Arsalani F, Narimani T, Azimian A, Akbari M. Detection of methicillin-resistance gene in Staphylococcus epidermidis strains isolated from patients in Al-Zahra Hospital using polymerase chain reaction and minimum inhibitory concentration methods. Adv Biomed Res 2013;2:23
|How to cite this URL:|
Pishva E, Havaei SA, Arsalani F, Narimani T, Azimian A, Akbari M. Detection of methicillin-resistance gene in Staphylococcus epidermidis strains isolated from patients in Al-Zahra Hospital using polymerase chain reaction and minimum inhibitory concentration methods. Adv Biomed Res [serial online] 2013 [cited 2020 May 27];2:23. Available from: http://www.advbiores.net/text.asp?2013/2/1/23/108008
| Introduction|| |
Coagulase-negative staphylococci (CNS) are considered important pathogens in nosocomial infections. About 80-90% of these bacteria are associated with nosocomial infections and show resistance to methicillin.  Staphylococcus epidermidis is a gram-positive and coagulase-negative bacterium which was initially considered a normal bacterial flora of healthy human skin and a commensal bacterium. In recent years, this bacterium has been known as the common cause of nosocomial infections.  These infections are mostly associated with using medical equipments such as intravenous and urinary catheters and joint shunts. S. epidermidis results in bacteremia, osteomyelitis, and peritonitis through these devices. ,
S. epidermidis is more virulent in immunosuppressed patients and other patients who are hospitalized for a long time.  Among the CNS, they cause 74-92% blood infections in hospitals.  Based on the studies conducted in western countries, more than 70% of S. epidermidis isolates are resistant to methicillin or oxacillin.  This bacterium produces biofilm and easily adheres to catheters and shunts and, through this mechanism, protects itself from the effect of antimicrobial agents. ,,,
The main compound of biofilm is cellular polysaccharide, which protects the bacterium against the body's immune system and also causes bacterial colonization on the surfaces of medical devices such as intravenous catheters and joint shunts, and creates resistance to external conditions.  It has been also proven that the bacteria inside the biofilm structure can easily exchange genetic information, such as antibiotic resistance genes, among themselves.  Resistance to methicillin in isolates which produce biofilm is considerably more than the other isolates which do not form biofilm.  Like Staphylococcus aureus, the mechanism of methicillin resistance occurs using the mecA gene which encodes penicillin binding proteins (PBPs) with little tendency to connect to the beta-lactam antibiotics. , This gene is located on chromosomal element named Staphylococcal Cassette Chromosome mec and is regulated by two other genes called mec1 and mecR1.  Concerns in detecting methicillin resistance are on the point that the sensitivity tests may not be able to identify correct resistance to methicillin. 
Methicillin resistance is identified by phenotypic and genotypic methods.  Nowadays, phenotypic methods such as disk diffusion are mostly used in laboratories, in which different environmental factors can affect bacterial growth and results.  Although several studies have shown that standardized disk diffusion method has similar sensitivity level to that of mecA gene, ,, some errors have also been reported. ,,, Therefore, it is essential to develop a rapid, sensitive, and accurate method to detect mecA gene, not affected by the conditions of the culture medium. In this study, we compared phenotypic (E-test) and polymerase chain reaction (PCR) genotypic methods to evaluate methicillin resistance in S. epidermidis. E-test method is derived from agar dilution and disk diffusion which has more advantages and requires less time compared to phenotypic methods. 
PCR is a rapid, sensitive, and accurate test for determination of mecA gene, and therefore methicillin resistance in these bacteria, and also used for confirming phenotypic methods which are less sensitive. 
Considering the high prevalence of S. epidermidis in various infections in infants, urinary infections, and its adherence to medical equipment and devices which leads to infection in different patients, and due to the considerable increase of methicillin resistance among patients, it was decided to compare the phenotypic and genotypic methods for evaluation of methicillin resistance in S. epidermidis.
| Materials and Methods|| |
A total of 146 S. epidermidis isolates were isolated from patients of different wards of Al-Zahra Hospital in Isfahan during 2009.
The collected isolates were identified by different conventional methods including gram staining, catalase test, tube test and slide coagulase test, DNAse, Novobiocin sensitivity, bacitracin and polymyxin B resistance, urea hydrolysis and Voges-Proskaer test, and finally culture in mannitol salt agar medium.
Antibiotic susceptibility test
Disk diffusion and minimum inhibitory concentration (MIC) determination with E-test method were performed for all isolates. Disk diffusion and MIC were accomplished according to the guidelines of Clinical and Laboratory Standards Institute (CLSI) (ref. CLSI). We used 30 μg oxacillin disk (HiMedia Code: SD088, India, Mumbai) for disk diffusion test. An E-test stripe was utilized for MIC determination, and S. aureus ATCC 25923 and ATCC 33591 was used as mecA negative and positive control, respectively.
Genomic DNA extraction
Genomic DNA was extracted by conventional phenol-chloroform method. 
Thermal cycling for amplification of mecA gene was performed in an Eppendorf thermal cycler (Mastercycler ® gradient). Amplification protocol consisted of 5 min initial denaturation at 94°C, followed by 35 cycles of denaturation (94°C/30 seconds), annealing (52°C/30 seconds), and extension (72°C/60 seconds), and an additional post-amplification extension step at 72°C for 5 min.
The following primers were used for PCR amplification of mecA gene: ,
PCR was performed in a mixture of 25 μl volume containing: 2.5 μl 10 × buffer (Roche Germany, Berlin), 0.4 μl of each dNTP (200 μm), 2.5 μl (50 mm) MgCl 2, 2.5 U of Taq DNA polymerase, 10 pmol of each primer, and 5 μl of template DNA.
| Results|| |
Of the total 146 S. epidermidis strains studied, 110 bacterial samples (75.34%) were methicillin resistant and contained mecA gene and 36 samples (24.66%) were methicillin sensitive not harboring mecA gene [Figure 1], [Figure 2], [Figure 3]. The specificity and sensitivity of E-test, disk diffusion, and PCR are compared in [Table 1]. Our results showed good correlation between phenotypic and genotypic methods for detection of antibiotic susceptibility.
|Table 1: Comparing sensitivity and specificity of PCR, E-test, and disk diffusion method |
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|Figure 1: Negative results of the tested bacteria (methicillin-sensitive) in E-test method|
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|Figure 2: Results of the tested bacteria (resistant to methicillin) in E-test method|
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|Figure 3: PCR results of mecA gene in 7 isolates staphylococcus epidermidis. Column 10: Size marker (50 bp). Column 8, 1-6: Isolates of staphylococcus epidermidis containing mecA gene. Column 7: Positive control of staphylococcus aureus ATC 33591. Column 9: Negative control of staphylococcus aureus ATCC 25923|
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| Discussion|| |
Methicillin resistance in isolates of CNS has increased significantly in the recent years. Approximately 50-80% of it depends on the species containing mecA gene or show resistance to oxacillin. Among the CNS isolates, Staphylococcus haemolyticus is the most frequent species in nosocomial infections and shows more resistance to oxacillin. 
Resistance to methicillin was reported shortly after using this drug for the treatment of staphylococcal infections in 1961, and then it spread to hospitals around the world.  According to the studies conducted on resistance to methicillin in CNS, especially S. epidermidis, in different countries in recent years, resistance to methicillin is not usually below 50%.  The reports presented have indicated that methicillin resistance is increasing worldwide, causing great concern. Some of these studies are discussed below. In a study conducted in the United States in 1994, approximately 80% of S. epidermidis strains isolated from nosocomial infections showed resistance to methicillin and most of these strains also had resistance to other antibiotics.  According to a study conducted in Finland, methicillin resistance in S. epidermidis increased from 28% in 1938 to 77% in 1994. 
In a report published by Oliveria et al. in Brazil in 2007, methicillin resistance in S. epidermidis was reported as 78.3%. 
According to a survey conducted in South Korea in 2001, resistance rates of CNS and S. epidermidis to methicillin was found to be 60-90%. 
In India, CNS resistance to methicillin was 20.8% between 1997 and 1998. 
During the present decade, resistance to methicillin has dramatically increased, causing various problems in the treatment.
In this study, among the 146 samples of S. epidermidis isolated from Al-Zahra Hospital in Isfahan, 110 cases carried the mecA gene or resistance to methicillin, which included 75.34% of all the samples.
From the results of the above-mentioned studies, it is seen that there is a correspondence between the present results and those of the above reports, which indicates that methicillin resistance in S. epidermidis is increasing worldwide.
Concerns in detecting methicillin resistance indicate that the available antimicrobial sensitivity tests are not able to detect this resistance correctly.  Identification of methicillin resistance includes phenotypic and genotypic methods.  Nowadays, the phenotypic methods such as disk diffusion are more used in the laboratories; different environmental factors affect the growth of bacteria and antibiogram results. 
Although several studies have indicated that standard method of disk diffusion is sensitive enough for detecting mecA-positive isolates, ,, some errors have also been reported by this method. ,,, Hence, there is a need for rapid, accurate, and sensitive method which is not affected by the conditions of the medium.
In this study, E-test phenotypic method and PCR genotypic method were used to identify mecA gene and methicillin-resistant S. epidermidis. The E-test method was derived from agar dilution and disk diffusion, which has more advantages in comparison with other methods and requires less time.  In the present study, the frequency of mecA gene in disk diffusion and E-test methods was found to be 61.64% and 70.54%, respectively. E-test method is a simple and cheap phenotypic test which is used for detecting methicillin resistance. This method was first presented in 1988 and then introduced in 1991 by a business company called AB Biodisk.
Ferreira et al. studied methicillin resistance in 132 isolates of CNS using disk diffusion, E-test, and PCR methods, and reported that the sensitivity and specificity of disk diffusion test were 94.2% and 91.8%, respectively, and those of E-test were 100% and 71.4%, respectively.
While reviewing new techniques for determining methicillin resistance, Swenson reported that phenotypic methods had high sensitivity although they did not reach 100%. He also reported that disk diffusion tests had low sensitivity in the range of 61-85%. ,,
Considering the results obtained in other studies and the results of the present work, it could be stated that disk diffusion, as a phenotypic test, had slower sensitivity and specificity in comparison to E-test.
According to [Table 1], in the false-negative and -positive phenotypic tests, it was observed that these results were more tangible in disk diffusion test.
Gerberding et al. and Chambers noted that the false-negative results were the heterogeneousness of mecA gene. ,
Chambers noted (in the same year) that the false-positive results were due to two factors of producing excessive penicillinase and great variation of PBPs. 
| Conclusions|| |
In this study, three methods, E-test, PCR, and disk diffusion, were used to study methicillin resistance in S. epidermidis isolates. It was found that PCR was more precise and accurate than the other two methods; moreover, phenotypic method of E-test was a cheap and simple method for evaluating methicillin resistance. The result of this study indicated that resistance to methicillin in S. epidermidis in Iran is rapidly increasing, similar to other countries and even the developed ones.
| References|| |
|1.||Forbes BA, Sahm DF, Weissfeld AS. Types of nosocomial infections. In: Shanahan JF, editor. Bailey-Scotts diagnostic microbiology. 12 th ed. Vol. 20. Philadelphia: Mosby; 2002. p. 68-9. |
|2.||Lim SM, Webb SA. Nosocomial bacterial infections in Intensive Care Units. I. Organisms and mechanisms of antibiotic resistance. Anaesthesia 2005;60:887-902. |
|3.||von Eiff C, Jansen B, Kohnen W, Becker K. Infections associated with medical devices: Pathogenesis, management and prophylaxis. Drugs 2005;65:179-214. |
|4.||Barrau K, Boulamery A, Imbert G, Casalta JP, Habib G, Messana T, et al. Causative organisms of infective endocarditis according to host status. Clin Microbiol Infect 2004;10:302-8. |
|5.||Ziebuhr W. Staphylococcus aureus and Staphylococcus epidermidis: Emerging pathogens in nosocomial infections. Contrib Microbiol 2001;8:102-7. |
|6.||Garrett DO, Jochimsen E, Murfitt K, Hill B, McAllister S, Nelson P, et al. The emergence of decreased susceptibility to vancomycin in Staphylococcus epidermidis. Infect Control Hosp Epidemiol 1999;20:167-70. |
|7.||Dickinson TM, Archer GL. Phenotypic expression of oxacillin resistance in Staphylococcus epidermidis: Roles of mecA transcriptional regulation and resistance-subpopulation selection. Antimicrob Agents Chemother 2000;44:1616-23. |
|8.||Hebert GA. Hemolysins and other characteristics that help differentiate and biotype Staphylococcus lugdunensis and Staphylococcus schleiferi. J Clin Microbiol 1990;28:2425-31. |
|9.||Hebert GA, Crowder CG, Hancock GA, Jarvis WR, Thornsberry C. Characteristics of coagulase-negative staphylococci that help differentiate these species and other members of the family Micrococcaceae. J Clin Microbiol 1988;26:1939-49. |
|10.||Kloos WE, Bannerman TL. Update on clinical significance of coagulase-negative staphylococci. Clin Microbiol Rev 1994;7:117-40. |
|11.||Mack D, Davies AP, Harris LG, Rohde H, Horstkotte MA, Knobloch JK. Microbial interactions in Staphylococcus epidermidis biofilms. Anal Bioanal Chem 2007;387:399-408. |
|12.||Vuong C, Voyich JM, Fischer ER, Braughton KR, Whitney AR, DeLeo FR, et al. Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system. Cell Microbiol 2004;6:269-75. |
|13.||Arciola CR, Campoccia D, Gamberini S, Donati ME, Pirini V, Visai L, et al. Antibiotic resistance in exopolysaccharide-forming Staphylococcus epidermidis clinica isolates from orthopaedic implant infevtions. Biomaterials 2005;26:6530-5. |
|14.||Koksal F, Yasar H, Samasti M. Antibiotic resistance patterns of coagulase-negative staphylococcus strains isolated from blood cultures of septicemic patients in Turkey. Microbiol Res 2009;164:404-10. |
|15.||Hiramatsu K, Cui L, Kuroda M, Ito T. The emergence and evolution of methicillin-resistance Staphylococcus aureus. Trends Microbiol 2001;9:486-93. |
|16.||Hiramatsu K, Katayama Y, Yuzawa H, Ito T. Molecular genetics of methicillin-resistance staphylococcus aureus. Int J Med Microbiol 2002;292:67-74. |
|17.||Chambers HF. Methicillin resistance in staphylococci: Molecular and biochemical basis and clinical implications. Clin Microbiol Rev 1997;10:781-91. |
|18.||York MK, Gibbs L, Chehab F, Brook GF. Comparison of PCR detection o mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci. J Clin Microbiol 1996;34:249-53. |
|19.||Martins A, Cunha Mde L. Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: Epidemiological and molecular aspects. Microbiol Immunol 2007;51:787-95. |
|20.||Mirsalehian A, Jabalameli F, Alizadeh S. Comarison of disk agar diffusion susceptibility testing and PCR in detection of methicillin resistance Staphylococcus aureus. Tabriz Univ Med J 2003;61:420-5. |
|21.||Hedin G, Lofdahl S. Detecting methicillin-resistant Staphylococcua epidermidis-disc diffusion, broth breakpoint or polymerase chain reaction? APMIS 1993;101:311-8. |
|22.||Olsson-Liljequist B, Larsson P, Ringertz S, Lodhal S. Use of a DNA hybridization method to verify results of screening for methicillin resistance in staphylococci. Eur J Clin Microbiol Infect Dis 1993;12:527-33. |
|23.||McDonald CL, Maher WE, Fass RJ. Revised interpretation of oxacillin MICs for Staphylococcus epidermidis based on mecA detection. Antimicrob Agents Chemother 1995;39:982-4. |
|24.||Ferreria RB, Iorio NL, Malvar KL, Nunes AP, Fonseca LS, Bastos CC, et al. Coagulase-negative staphylococci: Comparison of phenotypic and genotypic oxacillin susceptibility tests and evaluation of the agar screening test by using different concentrations of oxacillin. J Clin Microbiol 2003;41:3609-14. |
|25.||Hussain Z, Stoakes L, Lannigan R, Lango S, Noncekivell B. Evaluation of screening and commercial methods for detection of methicillin resistance in coagulase-negative staphylococci. J Clin Microbiol 1998;36:273-4. |
|26.||Perazzi B, Fermepin MR, Malimovka A, Garcia SD, Orgambide M, Vay CA, et al. Accuracy of cefoxitin disk testing for characterization of oxacillin resistance mediated by penicillin-binding protein 2a in coagulase-negative staphylococci. J Clin Microbiol 2006;44:3634-9. |
|27.||Rosser SJ, Alfa MJ, Hoban S, Kennedy J, Harding GK. Etest versus agar dilution fpr antimicrobiol susceptibility testing of viridans group streptococci. J Clin Microbiol 1999;37:26-30. |
|28.||DeGiusti M, Pacifico L, Tufi D, Panero A, Boccia A, Chiesa C. Phenotypic detection of nosocomial mecA-positive coagulase-negative staphylococci from neonates. J Antimicrob Chemother 1999;44:351-8. |
|29.||Vannuffel P, Laterre PF, Bouyer M, Gigi J, Vandercam B, Reynaert M, et al. Rapid and specific molecular identification of methicillin-resistant Staphylococcus aureus in endotracheal aspirates from mechanically ventilated patients. J Clin Microbiol 1998;36:2366-8. |
|30.||Caierao J, Superti S, Dias CA, d'Azevedo PA. Automated systems in the identification and determination of methicillin resistance among coagulase negative staphylococci. Mem Inst Oswaldo Cruz 2006;101:277-80. |
|31.||Stefani S, Varaldo PE. Epidemiology of methicillin-resistant staphylococci in Europe. Clin Microbiol Infect 2003;9:1179-86. |
|32.||Rupp ME, Archer GL. Coagulase-negative staphylococci: Pathogens associated with medical progress. Clin Infect Dis 1994;19:231-43. |
|33.||Martins A, Cunha Mde L. Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: Epidemiological and molecular aspects. Microbiol Immunol 2007;51:787-95. |
|34.||Oliveira AD, d'Azevedo PA, de Sousa LB, Viana-Niero C, Francisco W, Lottenberg C, et al. Laboratory detection methods for methicillin resistance in coagulase negative Staphylococcus isolated from ophthalmic infections. Arq Bras Oftalmol 2007;70:667-75. |
|35.||Nam MH, Woo HY, Lee JH, Lee NY. Comparison of mecA gene detection with susceptibility testing methods in coagulase negative Staphylococcus according to the new NCCLS guidelines. Korean J Clin Microbiol 2000;3:57-61. |
|36.||Sharma V, Jindal N, Devi P. Prevalence of methicillin resistant coagulase negative staphylococci in a tertiary care hospital. Iran J Microbiol 2010;2:185-8. |
|37.||Tveten Y, Jenkins A, Digranes A, Melby KK, Allum AG, Kristiansen BE. Comparison of PCR detection of mecA with agar dilution and E-test for clinical isolates of coagulase negative Staphylococci. Clin Microbiol Infect 2004;10:459-70. |
|38.||Swenson JM. News test for detection of oxacillin resistant Staphylococcus aureus. Clin Microbiol 2002;24:159-62. |
|39.||Gerberding JL, Miick C, Liu HH, Chambers HF. Comparison of conventional susceptibility tests with direct detection of penicillin-binding protein 2a in borderline oxacillin-resistant strains of Staphylococcus aureus. Antimicrob Agents Chemother1991;35:2574-9. |
[Figure 1], [Figure 2], [Figure 3]
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