Indian J Med Res 127, January 2008, pp 78-84
Comparison of phenotypic versus genotypic methods in the
detection of methicillin resistance in Staphylococcus aureus
K.M. Mohanasoundaram & M.K. Lalitha
Department of Microbiology, Christian Medical College & Hospital, Vellore, India
Received October 5, 2006
Background & objectives: Conventional methods to detect methicillin resistance in Staphylococcus
aureus are inadequate as expression of resistance is subject to environmental and conditional
expression of PBP2a antigen. The objective of the present study was to determine methicillin
resistance in S. aureus by conventional susceptibility (oxacillin disc diffusion and oxacillin MIC)
and molecular methods (PCR) and to evaluate latex agglutination test for the detection of PBP 2a
and to compare the results of these tests for its sensitivity, specificity and rapidity.
Methods: A total of 150 consecutive clinical isolates of Staphylococcus aureus received at the
Department of Microbiology, Christian Medical College, Vellore, were included. Oxacillin (1 mg)
disc diffusion and agar dilution method were used. The isolates were also subjected to latex
agglutination test for detection of PBP2a and multiplex PCR to detect mecA and femB genes.
Results: Of the 150 isolates, 33 were found to be MRSA by oxacillin disc diffusion. By MIC method,
13 per cent of the isolates had values 32 mg/ml, 6 per cent between 16-8 mg/ml and 2.7 per cent had
a value of 4 mg/ml; 100 per cent concordance was obtained between the oxacillin disc screening and
MIC methods. The latex agglutination showed positive reaction for all MRSA with only one MSSA
being falsely classified as MRSA. The specificity and sensitivity were 99 and 100 per cent respectively.
Test results were obtained within 15 min. By multiplex PCR, all 22 per cent of MRSA were positive
for mecA and femB genes and additionally one MSSA carried mecA gene. However, femB gene was
not found in 6 MSSA isolates. Specificity and sensitivity of PCR for mecA detection was similar to
latex agglutination test. PCR system required approximately five hours.
Interpretation & conclusions: Our findings showed that the conventional methods for detection of
methicillin resistance like disc screening, disc diffusion and MIC are cost-effective but time
consuming. Latex agglutination though expensive is rapid and can be a good preliminary screen
with high sensitivity and specificity. Multiplex PCR is a good confirmatory test though expensive.
Key words femB - latex agglutination - mecA - methicillin resistant Staphylococcus aureus - multiplex PCR
Staphylococcus aureus is one of the most common
bacteria encountered in the clinical practice. Despite
the introduction of effective antimicrobial agents and
improvements in hygiene, staphylococci have persisted
as important hospital and community pathogens1-3.
Methicillin-resistant Staphylococcus aureus (MRSA)
78
MOHANASOUNDARAM & LALITHA: COMPARISON OF VARIOUS METHODS TO DETECT MRSA
are significant pathogens that have emerged over the
past 30 years to cause both nosocomial and community
acquired infections4-8. There has been a steady increase
in the prevalence of MRSA in hospitals in the United
States over the years such that now approximately 25
per cent of nosocomial isolates of S. aureus are
methicillin resistant5. Indian literature shows that MRSA
incidence was as low as 6.9 per cent in 1988 and reached
to 24 and 32.8 per cent in Vellore9 and Lucknow10 in
1994, respectively and was of the same order in
Mumbai, Delhi and Banglore in 1996 and in Rohtak
and Mangalore in 199911. However, in some of the
centres, it was as high as 80 per cent and in India the
isolation varied from 20-40 per cent12.
The infections caused by MRSA are serious and
are difficult to treat. Only a few antimicrobial agents
are available for treatment of such infections and none
of these possesses ideal characteristics. The reports
from India suggest increasing incidence of MRSA9,10,13.
Hence accurate and rapid identification of MRSA in a
clinical specimen is essential for timely decisions on
isolation procedures and effective antimicrobial
therapy12, 14-17.
Expression of methicillin resistance in the clinical
laboratory setting is subject to environmental conditions
i.e., temperature, pH, incubation time, and salt
concentration in the medium 12,14,15 . Conditional
expression of PBP2a antigen may cause ambiguity in
susceptibility tests8. To complicate matters further
methicillin resistance is often expressed
heterogeneously, in that only 1 in 104 to 107 cells of the
population is phenotypically resistant9,12,16. These factors
emphasize the need to develop a rapid, standardized,
accurate and sensitive method for detection of
methicillin resistance in staphylococci which is not
dependent on growth conditions.
The conventional methods to detect MRSA in the
laboratory include oxacillin agar screen, disk diffusion
using one microgram oxacillin disk as well as oxacillin
MIC by agar or broth dilution methods. Numerous
approaches that improve the turn around time for the
detection of MRSA have been described. Polymerase
chain reaction (PCR) is considered the gold standard16,18.
It is rapid with a high degree of sensitivity and
specificity, but is expensive. A combination of primers
for genes responsible for coagulase activity (femB) and
methicillin resistance (mecA) would identify all the
isolates correctly and can be used to perform a single
step multiplex PCR14,16,19,20.
79
Denka-Seiken Co., Niigata, Japan has developed a
simple and rapid latex agglutination assay for the
detection of methicillin resistance, which makes use of
a specific monoclonal antibody directed towards the
PBP2a antigen21-24. The present study was undertaken
to compare the conventional methods of identification
of MRSA with the molecular method (multiplex PCR)
for its accuracy, sensitivity, specificity and rapidity and
also to evaluate a rapid screening method of MRSA
detection, viz, latex agglutination (LA).
Material & Methods
Bacterial isolates: A total of 150 consecutive isolates
of S. aureus from clinical specimens that were collected
between March 2002 and September 2003 in the
Department of Microbiology, Christian Medical
College, Vellore, were included in the study. All the
isolates were identified as S. aureus by culture and
biochemical tests which included test for clumping
factor, free coagulase and mannitol fermentation. The
biochemical tests were carried out as per the standard
procedure done routinely in our laboratory. All these
isolates were then subjected to phenotypic and
genotypic susceptibility testing. The phenotypic
methods included were oxacillin disc diffusion test,
oxacillin MIC and latex agglutination test for detection
of PBP2a antigen. The genotypic method17 done was
multiplex PCR for the detection of mecA and femB
genes. All the isolates were tested by disc diffusion
against a panel of antimicrobial agents which included
erythromycin, tetracycline, gentamicin, co-trimoxazole,
ciprofloxacin, ofloxacin, rifampicin and netilmicin to
compare their susceptibility pattern. Two standard
strains, one MRSA ATCC(43300) and one methicillinsensitive S. aureus (MSSA) ATCC (29213) were
included in each batch of testing by all methods.
Oxacillin disc diffusion test: Disc diffusion test was
performed on all isolates of S. aureus with 1mg of
oxacillin per disc on 25 ml of Mueller-Hinton agar
without NaCl supplementation. The zone of inhibition
was determined after 24 h of incubation at 37ºC. The
zone size was interpreted according to the National
Committee for Clincial Laboratory Standards (NCCLS)
criteria25: susceptible-13 mm; intermediate-11-12 mm;
and resistant - 10 mm.
MIC of oxacillin (agar dilution method): The MuellerHinton agar was used for all isolates of S. aureus. The
inoculum was prepared by emulsifying portions of 4-5
discrete colonies into 4-5 ml of nutrient broth to opacity
adjusted by McFarlands standard 0.5. An aliquot of
80
INDIAN J MED RES, JANUARY 2008
1 in 20 dilutions with normal saline (0.001 ml) was
used as the final inoculum. The concentrations of
oxacilin used were 32-0.015 mg/ml26.
mecA 2- 5´ CCA ATT CCA CAT TGT TTC GGT CTA
A 3´ (25 nucleotides) yielding 540 bp, and femB gene
was detected using the primers
The plates were dried before they were inoculated.
The inoculum was applied as a spot of about 5-8 mm in
diameter. A platinum loop calibrated to deliver 0.001
ml of inoculum was used. Inoculated plates were left
undisturbed until the spots of inoculum have dried. The
plates were then incubated for 24 h at 37ºC. The
endpoint was the concentration of the antibiotic
completely inhibiting the growth. The results were
reported as the MIC value in mg/ml: susceptible < 2,
and resistant > 4.
femB 1- 5´ TTA CAG AGT TAA CTG TTA CC 3´ (20
nucleotides)
Multiplex PCR for the detection of mecA and fem B
genes: The bacterial DNA was extracted using QIAGEN
DNA extraction kit (QIA amp DNA mini kit Hilden,
Germany) and the procedure was followed according
to the manufacturer’s instructions.
Five microlitres of the extracted DNA was
transferred to 20 ml of PCR amplification mix consisting
of 2.5 ml of PCR buffer, 2.5 ml of MgCl2, 1.25U of Taq
polymerase, 4 ml of DNTPs and 1 ml of each primer.
The primers (Genosys, Biotechnology, Thewoodlands,
Texas) used for the detection of mecA gene were:
mecA 1-5´ GAA ATG ACT GAA CGT CCG ATA A 3´
(25 nucleotides)
femB 2- 5´ ATA CAA ATC CAG CAC GCT CT 3´ (20
nucleotides) yielding a 700 bp PCR product (Fig.).
Latex agglutination test for detection of PBP2a: The
Latex agglutination test MRSA Screen, DenkaSeiken,
Tokyo, Japan) was done as follows:
A loop full of bacterial cells was suspended in 200
ml of extraction reagent 1 and subsequently lysed by
boiling for 3 min. After cooling to room temperature,
50 µl of extraction reagent 2 was added to 200 ml of the
lysate and mixed well. After 1 min of centrifugation at
1500×g, rpm, 50 ml of the supernatant was used for
testing agglutination with sensitized latex particles
(1 drop) and another 50 ml of the supernatant was tested
with the control latex particles (1 drop). The test card
was rotated for 3 min and the resulting agglutination
patterns were visually read and the results were
recorded. S. aureus ATCC 29213 was used as negative
control. The results were interpreted as follows:
Strong agglutination against a clear background----3+
Agglutination against a slightly turbid background--2+
Fig. Agarose gel electrophoresis for mecA (540 bp) and femB (700 bp) genes. Lanes: 1 WHO MRSA – ATCC 43300; 2 ATCC 29213 MSSA;
3, 4, 6 & 8 clinical isolates of MSSA; 5 Molecular weight ladder; 7, 9, and 10 clinical isolates of MRSA.
MOHANASOUNDARAM & LALITHA: COMPARISON OF VARIOUS METHODS TO DETECT MRSA
Slight agglutination against a turbid background----1+
Homogeneous white suspension with no visible
agglutination-Negative. Agglutination patterns of 1+ or
greater were considered positive, except if agglutination
was also seen with the control latex.
Results
A total of 150 consecutive S. aureus isolates from
clinically important sites of infection (31% from bone
and joint infections and 25% from infections of skin
Table I. Distribution of oxacillin MIC values among S. aureus
isolates (n=150)
MIC value of oxacillin ( mg/ml )
Number
Per cent
20
4
5
4
0
0
11
95
11
150
13.3
2.7
3.3
2.7
0
0
7.3
63.4
7.3
100
>32
16
8
4
2
1
0.5
0.25
0.125
Total
Table II. Antimicrobial susceptibility pattern of methicillin resistant
strains of S. aureus (MRSA) [n=150, 33 were MRSA]
Antibiotics
Tetracycline
Chloramphenicol
Gentamicin
Norfloxacin
Co-trimoxazole
Ciprofloxacin
Ofloxacin
Rifampicin
Netilmicin
Amikacin
Erythromycin
Susceptible
Resistant
Resistance %
6
27
4
0
1
1
28
26
23
8
5
27
6
29
33
32
32
5
7
10
25
28
82
18
88
100
97
97
15
21
30
80
85
81
and subcutaneous tissue) were subjected to disk
diffusion using 1µg oxacillin disc, agar dilution,
multiplex PCR for detection of mecA and femB genes
and latex agglutination.
Antimicrobial susceptibility pattern of S.aureus by
disc diffusion method revealed susceptibility in 78 per
cent of the isolates and resistance in 22 per cent. A high
level of resistance (>32 mg/ml) was noted for oxacillin
in 20 (13.3%) isolates, moderate level resistance (16-8
mg/ml) in 9 (6%) and low level resistance (<4 mg/ml)
in 4 (2.7%) by oxacillin break-point method (Table I).
High resistance was noted for erythromycin (85%),
tetracycline (82%), gentamicin (88%), norfloxacin
(100%), co-trimoxazole (97%), and ciprofloxacin
(97%).The isolates showed moderate resistance to
rifampicin (21%), and netilmicin (30.3%) and low rates
of resistance to chloramphenicol (18%) and ofloxacin
(15%) (Table II).
The MRSA showed a high level of resistance to all
antimicrobials in general in comparison to the MSSA.
Also most of the MRSA in this study were actually
resistant to many classes of antimicrobials at the same
time and thus qualify as multiply drug resistant
Staphylococcus aureus (MDR-MRSA).
A multiplex PCR to detect mecA and femB genes
was done for all the 150 isolates of S. aureus and it
revealed the presence of mecA and femB in all the
isolates of MRSA. However, one isolate of MSSA
revealed the presence of mecA gene having the potential
to become MRSA in future and 6 MSSA isolates did
not reveal the presence of femB genes.
All the 150 isolates of S. aureus were subjected to
latex agglutination test. All the 33 isolates of MRSA as
detected by oxacillin disc diffusion using 1 mg disc and
PCR (mecA gene) showed positive (4+) reaction by latex
agglutination. Of the 117 isolates of MSSA, 116 were
Table III. Comparison of results of susceptibility testing methods with mecA gene analysis of S. aureus (n=150) and LA test
Oxacillin disc diffusion
(1 µg)
Pos
117
Neg
33
Agar dilution MIC
(µg/ml)
<2
117
>4
33
Latex agglutination
(LA) test
Pos
34
Neg
116
PCR result
mecA
Pos
34
femB
Neg
116
Pos
144
Neg
6
Table IV. Comparison of the different parameters between the tests
Method
Oxacillin disc diffusion (1mg)
Agar dilution (MIC)
Latex agglutination
PCR
Speed (h)
Cost (Rs)
48-72
48-72
15 min
5
20
13
300
600
Sensitivity (%)
97
97
100
100
Specificity (%)
100
100
99
100
82
INDIAN J MED RES, JANUARY 2008
negative by LA, but one isolate showed a 2+ reaction
(Table III). The results were confirmed by repeating
the latex agglutination procedure.
required for methicillin resistance in staphylococci. It
is essential for expression of methicillin resistance in
true MRSA strains6, 9, 12, 14.
All the tests were compared for different parameters
like speed, cost, sensitivity and specificity with PCR
for mecA gene, which revealed 100 per cent sensitivity
and specificity for PCR, and 100 per cent sensitivity
and 99 per cent specificity for latex agglutination, and
97 per cent sensitivity with 100 per cent specificity for
oxacillin screen and agar dilution methods. PCR and
latex agglutination tests were rapid when compared to
conventional methods, but at the same time very
expensive (Table IV).
Our study also revealed that one of the 117 MSSA
isolates when subjected to PCR revealed the presence
of mecA gene and the remaining 116 MSSA were found
to be mecA negative. This isolates namely, mecA positive
but probably non-PBP-2a producing has been referred
to as cryptically methicillin resistant20.
Discussion
Staphylococcus is a known pyogenic coccus
responsible for various suppurative and pus forming
diseases. It has a wide distribution and carriage rate
among the hospital personnel as well as the patients4,10.
The incidence of MRSA have been reported to be on
the rise; the value ranges from 5 to 50 per cent in
different institutionalized studies10. The incidence of
MRSA infections in Indian hospitals has been recorded
at between 18.4 and 33.5 per cent among hospitalized
patients26.
The antimicrobial susceptibility test was performed
for all 150 isolates of S. aureus by disc diffusion using
1 mg oxacillin disc and oxacillin MIC using agar dilution
methods. The results obtained revealed 100 per cent
concordance. Susceptibility test profile revealed a high
level of resistance amongst the S. aureus to most of the
commonly used antimicrobials. The results were
comparable to those in a previous study carried out in
our department9,26.
The conventional MRSA detection assays are
simple and relatively cheap methods for detecting
methicillin resistance. The sensitivity and specificity
of the conventional methods were found to be 97 and
100 per cent respectively. Accurate determination of
methicillin resistance in staphylococci by conventional
tests is subject to variations in inoculum size, incubation
time, medium pH, medium salt concentration, etc.
However, difficulties occur when organisms have their
MICs near the break-point. It is in such instances that
detection of mecA gene is useful by molecular technique.
In this study the PCR method revealed mecA positivity
in all 33 isolates which were resistant to oxacillin by
disc diffusion and break-point MIC method which
shows that the mecA gene is the primary structural gene
From a clinical perspective, it is important to
differentiate isolates that have mecA positive resistance,
which is the classic type of oxacillin resistance, from
the infrequently encountered isolates that have one of
the other types of more subtle or borderline resistance
due to hyperproduction of beta-lactamases1.
Isolates that possess the mecA gene are either
heterogenous or homogenous in their expression of
resistance. With homogenous expression, virtually all
cells express resistance when tested by standard in vitro
tests. However, testing of hetero-resistant isolates may
result in some cells that appear susceptible and others
resistant. Often only 1 in 104 to 1 in 108 mecA positive
cells in the test population expresses resistance and
heterogeneous expression results in MICs that appear
to be borderline. The clinical problem posed by such
isolates is that during chemotherapy with beta-lactam
antibiotics, production of PBP-2a may be induced
converting them into oxacillin resistant strains. For this
reason, detection of mecA gene is indispensable for
precise differentiation of MRSA and thus the PCR is a
useful technique in clinical laboratories.
The multiplex PCR used to detect femB gene
revealed its presence in all the 33 isolates of MRSA,
and 6 MSSA isolates did not reveal the presence of femB
genes even though these were positive for free coagulase
and mannitol fermentation tests. Kobayashi et al20 have
reported that though femB genes are detectable only in
S. aureus, an absence of femB gene does not mean that
the isolate is not S. aureus. They have shown that up to
3 per cent of S. aureus can give negative results for
femB genes20. Another interesting fact that was brought
to light in this study and which is consistent with the
findings of previous studies by Kobayashi et al20 is that
the femB negativity amongst S. aureus was mainly seen
in MSSA.
While comparing the findings of the conventional
methods of detection of MRSA with that of molecular
methods, it was noticed that the multiplex PCR
MOHANASOUNDARAM & LALITHA: COMPARISON OF VARIOUS METHODS TO DETECT MRSA
(co-amplification of both mecA and femB genes) was a
very specific and sensitive method. The PCR technique
has many added advantages over the conventional
techniques. The multiplex PCR can furnish results
within 24 h as compared to a minimum of 48 h required
by the conventional techniques. Detection of mecA gene
confirms the MRSA status of the isolate. Therefore,
PCR can be considered the gold standard for detection
of methicillin resistance.
The false negative results of the conventional
methods can be picked up by the PCR in very early
stage of the disease. One false negative result by the
conventional method and mecA positivity by PCR shows
the inherent potential of the isolate to develop high
levels of résistance to methicillin in the future. The
critical parameters for success of a PCR based test are
cost, reliability, speed, accuracy, and sensitivity.
All the 33 MRSA tested by oxacillin screen using
1µg disc and PCR (mecA) showed 4+ reaction in latex
agglutination test. However, of the 117 isolates of
MSSA, 116 were negative by LA method and one isolate
showed a 2+ reaction (false positivity). Even upon
retesting, the isolate gave a 2+ reaction. Cavassini et
al22 have reported that correct inoculum is important
since false-positive agglutination reactions occur when
the inoculum used was 10 times heavier than that
recommended. In this study the recommended inoculum
was used and the reason for false positivity (2+) could
not be identified. There are chances of false positivity
when the test card is rotated for more than 3 min.
Technically this test was easy to perform, requiring only
micro tubes, dry bath, a table centrifuge and a manual
pipetter. The test gave results within 15 min, making it
the fastest test to our knowledge to reliably detect
oxacillin resistance in S. aureus isolates.
The PCR and the latex agglutination tests have the
major advantage over the other phenotypic methods of
not being influenced by the various levels of expression
of the resistance, a parameter (incubation temperature,
salt concentration and incubation time) which in highly
heterogeneous resistant isolates tends to render classical
methods less accurate.
In conclusion, the conventional methods for detection
of methicillin resistance viz., disc diffusion and MIC are
cost-effective but time consuming. The latex agglutination
though expensive, is very rapid and can serve as a good
preliminary screen with high sensitivity and specificity.
Multiplex PCR is a good confirmatory test, but its use
could be limited to reference laboratories due to its cost.
83
Acknowledgment
The authors acknowledge the CMC FLUID Research Grant
and Indian Council of Medical Research (ICMR) for funding the
project.
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Reprint requests: Dr K.M. Mohanasoundaram, Assistant Professor, Department of Microbiology, IRT Perundurai Medical College
14, 3rd Street, Kandaiyan Thottow, Malligai Nagar, Soolai, Erode 638 004, India
e-mail: [email protected]