Advances in Infectious Diseases, 2013, 3, 44-49 Published Online March 2013 (
Rapid Identification of Methicillin Resistant
Staphylococcus aureus Using Real Time PCR*
Said Abbadi1,2#, Hamdy Youssef1, Dalal Nemenqani3, Ahmed S. Abdel-Moneim1,4
1Microbiology Department, College of Medicine, Taif University, Al-Taif, Saudi Arabia; 2Microbiology Department, Faculty of
Medicine, Suez Canal University, Ismailia, Egypt; 3Pathology Department, College of Medicine, Taif University, Taif University,
Al-Taif, Saudi Arabia; 4Virology Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt.
Received December 6th, 2012; revised January 8th, 2013; accepted February 1st, 2013
Screening for colonization with methicillin resistant Staphylococcus aureas (MRSA) is a key aspect of infection control
to limit the nosocomial spread of this organism. Current methods for the detection of MRSA in clinical microbiology
laboratories using conventional methods is time consuming. In this research we are trying to evaluate the use of real
time PCR for the detection of MRSA. The PCR assay was evaluated in clinical isolates of MRSA (n = 45) and methicil-
lin susceptible Staphylococcus aureas MSSA (n = 10). The diagnostic values of the assay showed high sensitivity and
specificity. This real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine
microbiological laboratory. Real-time PCR now is available in all laboratories so its use in identification of MRSA will
help in shortening the period for MRSA identification and will help in the success of infection control programs in hos-
Keywords: Diagnosis; MRSA; Rapid Detection; Real-Time PCR; Staphylococcus aureus
1. Introduction
Staphylococci are ubiquitous colonizers of human or ani-
mal skin and mucous membranes causing a variety of
syndromes. Staphylococcus aureus can cause nosocomial
and community-acquired infections ranging from mild
conditions, such as skin and soft tissue infections, to se-
vere, life-threatening sepsis [1]. Strains of methicillin-
resistant S. aureus (MRSA) were first detected in the
early 1960s, shortly after methicillin came into clinical
use [2]. During the past decade, an increase in the num-
ber of Methicillin resistant Staphylococcus aureus (MRSA)
cases has been widespread globally among healthy com-
munity [3]. The mechanisms responsible for this resis-
tance are diverse, and hundreds of resistance genes have
been characterized in both Gram-negative and Gram-
positive species. A core function of all bacteriology labo-
ratories is to determine the antibiotic susceptibilities of
bacterial isolates and to detect the resistance phenotypes
conferred by these resistant genes. If a panel of agents in
a particular antibiotic class is tested, the interpretative
reading of the resulting patterns of resistance or suscepti-
bility often suggests the underlying resistance mecha-
nisms, predicts second-line drugs to be tested and pro-
vides extended knowledge for decisions in antimicrobial
therapy as well as infection control. Methicillin resis-
tance in Staphylococcus is mainly mediated by the over-
production of PBP2a, an additional modified penicillin
binding protein (PBP) with low affinity for β-lactam an-
tibiotics. The mecA gene, the structural determinant that
encodes PBP2a, is therefore considered as a useful mo-
lecular marker of putative methicillin resistance in S.
aureus [4]. The mecA gene can be detected by PCR,
which is considered the gold-standard test to identify
methicillin resistance [5]. The results of the conventional
culture-based screening methods with cultivation, identi-
fication and antimicrobial susceptibility testing take 48 to
72 hr. Rapid detection of a specific resistance mechanism
in a molecular test allows clinicians avoiding potentially
inappropriate treatment options. To rapidly detect methi-
cillin-resistant Staphylococcus aureus (MRSA) nasal co-
lonization in patients, clinical microbiology laboratories
should select either PCR methods or selective agar-
based methods. Several chromogenic and differenttial
MRSA selective agars have been shown to yield results
within 18 to 24 hr [6]. In contrast, PCR methods can
yield results in 2 to 3 hr. Antibiotic resistance genes pro-
vide important targets for molecular detection techniques
[7]. Although molecular assays provide a quick identifi-
cation of MRSA colonization as the results of the test can
*No conflict of interest to declare.
#Corresponding author.
Copyright © 2013 SciRes. AID
Rapid Identification of Methicillin Resistant Staphylococcus aureus Using Real Time PCR 45
be available within a few hours, these methods are ex-
pensive. However, quick MRSA screening tests prevent
unnecessary procedures as well as the spread of MRSA
in case of colonized patients and thus reducing the over-
all hospital costs. It should be noticed that molecular
biological techniques, such as DNA sequencing, or the
use of DNA microarray technology [3], could provide
more accurate identification and classification tool, but
such techniques are difficult to apply in a routine clinical
laboratory. A rapid, conventional and automated identi-
fication method based on phenotypic characters is a more
practical approach for the daily clinical laboratory pro-
The current study aimed to evaluate the use of real
time PCR in detection of methicillin resistant Staphylo-
coccus aureus (MRSA) and to trace the dynamics of ge-
notypic changes and the shifts in the levels of suscepti-
bility to antibiotics of MRSA isolated from patients in
King Abdel Aziz Hospital, Al-Taif, Saudi Arabia during
the period from Jan. to Jun. 2012.
2. Materials and Methods
2.1. Bacterial Isolates
Fifty five strains of S. aureus were collected during the
period from Jan. to Jun. 2012 from different departments
of King Abdel Aziz Hospital, Al-Taif, Saudi Arabia. The
strains were tested phenotypically and genotypically for
the detection of MRSA. Thirty strains were collected
from surgical wounds, 7 from sputum, 6 from blood, 5
from nasal swabs, 5 from skin and 2 from abdominal
fluid. All isolates were identified according to colonial
and microscopical morphology, catalase and coagulase
production. The 55 strains were tested also by real-time
PCR to identify MRSA isolates.
2.2. Culture Conditions and Media
The clinical isolates were first plated onto Columbia
blood agar plates and incubated at 37˚C for 24 h. Identi-
fication of the MRSA strains was performed by standard
procedures including colony morphology, catalase reac-
tion and coagulase activity [8].
2.3. Antimicrobial Susceptibility Test
2.3.1. Detection of Oxacillin Resistance by Phenotypic
Characteristic colonies that were identified as S. aureus
were tested for oxacillin resistance by using Mueller-
Hinton agar and Disc-diffusion test according to NCCLS
guidelines [9].
2.3.2. Antimicrobial Susceptibility Test to Other
Antimicrobial susceptibility test to a range of antimicro-
bial agents was performed using the following discs (aug-
mentin, cotrimoxazole, fusidic acid, gentamicin, erythro-
mycin, ciprofloxacin, clindamycin, amikacin, cepha lo-
thin, linezolid, teicoplanin and vancomycin). The pro-
cedure was performed by adopting the Kirby-Bauer disc
diffusion method using Muller-Hinton broth and agar and
antibiotic discs according to NCCLS guidelines [9].
2.3.3. Detection of Oxacillin Resistance by Genotypic
Method DNA Extraction
DNA was extracted from different bacterial isolates us-
ing DNA extraction kit (Koma Bioteck Inc., Korea).
Briefly, the bacterial pellet from each isolate was re-sus-
pended in 200 μl lysozyme reaction solution (20 mg/ml
lysozyme, 20 mM Tris-HCl pH 8, 2 mM EDTA, 1.2%
triton) and incubated for 30 min at 37˚C. The sample was
then incubated with proteinase K 20 µl and lysis buffer
200 µl at 60˚C for 30 min. and then further incubated at
95˚C for 15 min. Absolute ethyl alcohol 200 µl was then
added to each sample, vortexed then loaded into XPTG
mini column in a collection tube. The tubes were centri-
fuged at 13,000 rpm for 1 min. and the flow was dis-
carded. The column was then washed twice then dried
for 3 min at 13,000 rpm before the DNA was eluted in
new clean sterile tube using 100 µl of sterile double dis-
tilled water. Real-Time PCR
The real-time PCR assay was performed using comer-
cial TaqMan hydrolysis probe based MERSA real time
PCR detection kit (Liferiver, Shanghai, China) in Ep-
pendorf Mastercycler® eprealplex. The detection of the
amplified MERSA DNA amplicon was performed in
fluorimeter channel FAM with the fluorescent quencher
BHQ1. Amplification reactions were performed accord-
ing to the in a volume of 25 µl containing 2.5 µl of DNA
template, 21.5 µl reaction mix, 0.4 µl enzyme mix, 1 µl
internal control according to the manufacturer’s instruc-
tions. The cycling conditions included: 37˚C for 2 min,
an initial denaturation step at 94˚C for 2 min followed by
40-cycles consisted of heating at 93˚C for 15 sec and
annealing/elongation step at 60˚C for 1 min.
3. Results
Fifty five strains of S. aureus were collected during the
period from Jan. to Jun. 2012 from different departments
of King Abdel Aziz Hospital, Al-Taif, Saudi Arabia.
Thirty strains were collected from surgical wounds, 7
from sputum, 6 from blood, 5 from nasal swabs, 5 from
skin, and 2 from abdominal fluid as shown in (Table 1).
The most frequent isolation of the S. aureus in relation to
the age was noted in the age group of 21 - 40 years
(40%), followed by those in the age group of 41 - 60
Copyright © 2013 SciRes. AID
Rapid Identification of Methicillin Resistant Staphylococcus aureus Using Real Time PCR
Table 1. Number of identified S. aureus and the percent of
the resistant and susceptible of S. aureus to methicillin and
oxacillin (by disc diffusion method).
No. of Isolated
S. aureus MRSA MSSA
Skin 5
4 1
Nose 5
3 2
Blood 6
4 2
Wounds 30
27 3
Sputum 7
6 1
Abdominal fluid 2
1 1
Total 55 45 10
years (35%), >60 years (16%) and 0 - 20 years (9%) re-
spectively (Table 2). A total number of 45 isolates (82%)
were identified as MRSA while 10 isolates were MSSA.
Table 3 shows the antimicrobial resistance patterns of
MRSA and MSSA strains. Glycopeptides and linezolid
exhibited excellent activity against both MRSA and
MSSA. The other antibiotics which were found to be
effective against MRSA were amikacin (90.2%) and azi-
theromycin (78.2%). The overall drug resistance was
more in MRSA isolates in comparison to MSSA. As
many as 24% MRSA strains were resistant to multiple
drugs in comparison to 10% of MSSA.
3.1. Detection of MRSA by Phenotypic Methods
Forty five strains (82%) out of 55 tested strains were re-
sistant phenotypically to methicillin and oxacillin which
was confirmed by real time PCR.
3.2. Evaluation of the PCR with Clinical Isolates
All of the 55 clinical isolates of MRSA were detected by
real time PCR. The melting curve analysis showed a Tm
of 60˚C (Table 4 and Figure 1). All other clinical iso-
lates of MSSA (10 strains) were tested negative by real
time PCR.
4. Discussion
S. aureus is a major pathogen causing a wide spectrum of
clinical manifestations and beta-lactam antibiotics are the
drugs of therapeutic choice. Since the introduction of
methicillin into clinical use on 1961, the occurrence of
MRSA strains has enormously increased and MRSA is
now one of the most important nosocomial pathogens
worldwide [10]. Infections caused by MRSA require
treatment with glycopeptide antibiotics, which are ex-
pensive and may have serious side effects. Therefore,
rapid differentiation of MRSA strains from MSSA has
important implications for the treatment but also for the
Table 2. The frequency of Staphylococcus aureus isolation in
relation to the age group.
Age group (years) Number of isolation Percentage
0 - 20 5 9
21 - 40 22 40
41 - 60 19 35
>60 9 16
Total 55 100
Table 3. Antimicrobial susceptibility patterns among ( MR SA )
and (MSSA) strains.
Percentage of
susceptible MRSA
Percentage of
susceptible MSSA
Ciprofloxacin 30 85
Gentamicin 36 68
amikacin 90.2 98.6
Azithromycin 78.2 82
Clindamycin 66.0 78.0
Cotrimoxazole 41.4 64.8
Fusidic acid 74 86
Erythromycin 44.6 59.4
augmentin 18.6 56.3
Cephalothin 14.0 62
Linezolid 100 100
Teicoplanin 100 100
Vancomycin 100 100
Table 4. Comparison of MRSA detection results for clinical
isolates by culture identification and real time PCR.
Identification of StaphylococciNumber of isolates tested by PCR
By conventional methods Total +ve e
S. aureus, Oxacillin resistant 45 45 0
S. aureus, Oxacillin susceptible10 0 10
Total 55 45 10
management of patients with MSSA infections since ex-
tensive isolation measures is taken to limit the spread of
MRSA [11]. Regarding the age of the patients, we found
the occurrence of S. aureus to be higher among patients
in the age group 21 - 40 years (Table 2). Mulla, et al.,
[12] also reported that S. aureus was commonly isolated
from patients in age group 21 - 30 years. MRSA has be-
come prevalent with the discriminate and overuse of an-
Copyright © 2013 SciRes. AID
Rapid Identification of Methicillin Resistant Staphylococcus aureus Using Real Time PCR
Copyright © 2013 SciRes. AID
Figure 1. Real Time PCR results of some MRSA strains used in this study.
MRSA strains are usually introduced into aninstitution
by a colonized or infected patient. Rapid screening on
admission of high-risk patients for MRSA is one of the
control measures to limit the spread of MRSA strains.
Several studies have found that such screening programs
are cost-effective [16,17]. However, in the clinical labo-
ratory, conventional culture methods are time-consuming
and misinterpretation occurs sometimes when methicillin
resistance is simulated by the hyper production of beta-
lactamase [5] or coagulase activity is weak [18].
tibiotics. MRSA is of great concern not only because of
its resistance to methicillin, but also because it is gener-
ally resistant to many other commonly used antimicrobi-
als. Many studies have reported an increased in the inci-
dence of MRSA during recent years [13]. Our study
shows that MRSA was found to be 82% in consistent
with other data [14]. In our study, it was noted that the
sensitivity to most of antibiotics tested among MSSA
was significantly higher than MRSA. In our study, ami-
kacin sensitivity among the S. aureus strains was still
high in comparison to the findings from other countries
[15]. The sensitivity to amikacin was noted in 90.2% and
98.6% of MRSA and MSSA strains respectively. Our
study showed that there was an increase in the rate of
incidence of MRSA, which showed 100% susceptibility
to vancomycin, linezolid and teicoplanin. Upon compare-
ing our results of antibiotic susceptibility with other re-
sults inside Saudi Arabia, we found that all results (Ta-
ble 3) in consistent with the results of Moussa and Hes-
san [15] except for ciprofloxacin in which our results
showed only 30% of MRSA isolates were susceptible in
comparison to 60% in their results.
Based on these arguments, rapid and reliable methods
for the detection of MRSA are required. The identifica-
tion of MRSA strains based on the detection of the mecA
gene and simultaneous detection of an S. aureus speci-
fic marker gene by molecular methods has been intro-
duced as an interesting tool in clinical laboratories [7,
19,20]. In comparison to conventional PCR protocols
with the detection of the PCR products on gel electro-
phoresis, real-time PCR assays are time-saving. All the
presented assays, however, risk of co-amplifying the S.
aureus specific marker gene together with the mecA gene
from methicillin resistance coagulase negative staphylo-
Rapid Identification of Methicillin Resistant Staphylococcus aureus Using Real Time PCR
cocci (MRCNS), leading to false-positive results. In cli-
nical samples and consequently in enrichment broths
MSSA and MRCNS could simultaneously be present.
In the present study, we reported the use and evalua-
tion of a real-time PCR assay for MRSA screening from
clinical isolates. The use of this new method together
with a fast DNA extraction protocol to screen clinical
isolates achieved a sensitivity of 100% (45 of 45 detected
MRSA strains) and 100% specificity (no false-positive
result). Results are available within 90 min and the total
hands-on time was about 30 min.
The power of the PCR technique described here was
confirmed by another study performed by Huletsky et al.
[3]. Using five different forward primers in combination
with three different probes and one reverse primer they
were able to show that the vast majority of a world-wide
collection of MRSA isolates was detected by their PCR
assay. The sensitivity of their PCR strategy was 98.7%
compared to 100% in the current study.
The real-time PCR assay used in the current study
proved to be a sensitive and reliable tool for a rapid iden-
tification of MRSA from isolated colonies. The use of
PCR technology is more expensive than the use of con-
ventional methods. The clinical and economic benefit of
saving time in regard to expenses remains to be eluci-
dated. According to the hygiene policy in some countries,
patients highly suspected to be colonized with MRSA are
isolated in a single room until cultures become MRSA
negative. An earlier MRSA negative report in such cases
by a quick PCR assay as presented in this study will help
to reduce isolation time and costs.
5. Conclusion and Recommendation
Our real-time PCR assay proved to be a sensitive andre-
liable tool for the rapid identification of MRSA fromiso-
lated colonies. The use of PCR technology is more ex-
pensive than the use of conventional methods. The clini-
cal and economic benefit of saving time in regard to ex-
penses remains to be elucidated. According to the hy-
giene policy in some countries, patients highly suspected
to be colonized with MRSA are isolated in a single room
until cultures become MRSA negative. An earlier MRSA
negative report in such cases by a quick PCR assay as
presented in this study will help to reduce isolation time
and costs.
6. Acknowledgements
This study was supported by a research grant (Project No.
1-33-1848) through the Research Support of Taif Uni-
versity, Saudi Arabia.
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