Advances in Infectious Diseases, 2012, 2, 82-88
http://dx.doi.org/10.4236/aid.2012.23013 Published Online September 2012 (http://www.SciRP.org/journal/aid)
Heterogeneity in femA in the Indian Isolates of
Staphylococcus aureus Limits Its Usefulness as a Species
Specific Marker
Rosy Chikkala1, Nikhil Oommen George1, Kamaraju S. Ratnakar2, Ranganathan Natarajan Iyer3,
Venkataraman Sritharan1*
1Molecular Diagnostics and Biomarkers Laboratory, Department of Laboratory Medicine, Global Hospitals, Hyderabad, India;
2Global Hospitals, Hyderabad, India; 3Department of Clinical Microbiology, Global Hospitals, Hyderabad, India.
Received May 31st, 2012; revised July 1st, 2012; accepted August 3rd, 2012
ABSTRACT
Increase in prevalence of MRSA worldwide and hence the need for rapid detection, have led to use of molecular meth-
ods for confirmation of the species and also MRSA. Species specific markers like fem or nuc along with the methicil-
lin-resistance determinant, mecA, have been used by several investigators worldwide for the identification of MRSA. In
the current study, we have screened 54 microbiologically confirmed (MRSA, MSSA and CoNS) isolates for the pres-
ence of mecA, 16S rRNA, femA and nuc markers. While mecAPCR and 16S rRNAPCR results were consistent with
other studies, femA and nuc showed dramatic variation in detection rate (sensitivity) of S. aureus 29.6% and 53.7% re-
spectively. Evidences are presented to demonstrate the absence of femA. Our attempt to amplify the complete femA gene
using sequences flanking femA further confirmed these results and also indicated that variations exist even in the ge-
nomic sequences around femA. Our data reveals the need for exercising care while using primers designed on se-
quences of constitutive genes like femA and nuc for PCR based identification of S. aureus species. Though geograph ic
variations in the genome of S. aureus have previously been reported from around the world, we present here evidence
for the first time from India for absence of femA and also for prob able var iations in the seq uences around the femA gene
in clinical isolates of S. aureus.
Keywords: femA; mecA; nuc; PCR; S. aureus
1. Introduction
Staphylococcus aureus is one of the major causes of wide
spread gram positive bacterial nosocomial infections,
especially the post-surgical wound infections [1]. Its d is-
ease manifestations range from minor skin infections to
life-threatening diseases such as pneumonia, sepsis etc.
The emergence of MRSA in patients with no apparent
risk factors seems to be a growing concern. In India the
prevalence of MRSA is 51.8% in 20 10 [2]. Although the
discovery of penicillin proved to be a major breakthrough
in treating these infections, with it had also emerged a
major concern; the notorious ability of Staphylococcus
aureus to develop resistance to antibiotics and remain non-
responsive to treatment [3]. Shortly after the introduc tion
of methicillin, reports of Methicillin-resistant Staphylo-
coccus aureus (MRSA) had begun to surface within hos-
pitals in the early 1960s, which are now increasingly pre-
valent worldwide [4,5]. Such endemic MRSA infections
are difficult to eradicate and remain active reservoirs of
infection thereby increasing the hospital costs, length of
hospital stays, morbidity and mortality.
Methicillin-resistance in staph ylococci is expressed b y
the mecA gene that produces a “Penicillin Binding Pro-
tein 2a” (PBP2a) [6], a modified transpeptidase. This
PBP-2a has lower affinity to penicillin and its derivatives
than the other PBPs [7]. The Staphylococcal Chromo-
some Cassette mec (SCCmec), a mobile genetic element,
is composed of the mec gene complex which includes
mecA, its regulatory genes mecI, mecR and a ccr gene
complex. The latter encodes site specific recombinases,
namely ccrA/B, which in-turn regulate the mobility of
SCCmec [8]. Significant geographic variations have been
found in the structural organization of the SCCmec and
these variations have been used to classify the SCCmec
types [9]. However, mecA alone does not solely confer
the methicillin resistance. Studies have shown that fem
(factors essential for methicillin-resistance) or the auxiliary
genes like fem A/B/X in addition to mecA are also impor-
*Corresponding a uthor.

Heterogeneit y in femA in the Indian Isolates of Staphylococcus aureus Limits Its Usefulness
as a Species Specific Marker 83
tant in the expression of methicillin resistance [10]. The
femABX operon encodes factors which are responsible
for the formation of pentaglycine bridges in the cell wall
of Staphylococci [11].
Kirby-Bauer antibiotic testin g (disc diffusion antibiotic
sensitivity test), oxacillin agar screen test, agar dilution
and the Epsilometer test (E-test) are commonly used to
determine the MRSA phenotype in the clinical microbe-
ology laboratory. These tests have limitations and fre-
quently show variations [12]. Although the detection of
mecA gene remains the gold standard for detecting me-
thicillin-resistance, its detection alone does not confirm
the presence of Staphylococcus aureus [13] and there is
no consensus on the molecular target that could be used
to confirm the S. aureus species. Constitutively expressed
genes such as femA, femB and nuc are being used as mo-
lecular targets for the identification of Staphylococcus
aureus [14,15]. Polymorphisms within these constitutive
genes have been reported [16] worldwide and also fail-
ures to confirm the species of S. aureus using these genes
as targets for PCR detection [17]. However, from India,
there are no reports about any variations in the sequences
of these co ns titu tive g en es ye t and for th at matter SCCmec
or the genome of S. aureus are yet to be characterised in
India. The aim of our study was to optimise and estab lish
a multiplex PCR protocol to screen clinical isolates for
molecular markers of MRSA namely, 16S rRNA, mecA,
femA or nuc. We are reporting here absence of femA gene
in several clinical isolates of S. aureus (both MRSA and
MSSA), poor sensitivity of nuc as a target for S. aureus
identification and probable variation in the sequences
flanking the femA. These findings have serious implica-
tions for molecular diagnosis of S. aureus and are reflec-
tive of the geographic changes occurring in the gen o me o f
S. aureus.
2. Materials and Methods
54 isolates of S. aureus were collected from Jan 2012 to
March 2012 in Global Hospital, Hyderabad. These strai ns
were isolated on Mueller Hinton Agar (MHA) either
from blood culture bottles or directly from different
clinical specimens (pus, drain fluids/secretions etc). Co-
agulase and catalase tests were used to confirm S. aureus
in the isolates. Standard disc diffusion test usin g the anti-
biotic oxacillin were used to assess th e drug sensitiv ity o f
these isolates. Epsilometer test (E-test) was used to find
out the MIC of the isolates.
2.1. Preparation of Bacterial DNA Lysates
A single bacterial colony was scrapped off the agar and
the cells washed by suspending in 100 µl of TEX buffer
(10 mM Tris-HCl pH 8.5, 1 mM EDTA, 1% (w/v) Triton
X-100). The suspension was vortexed to achieve a uni-
form suspension and was centrifuged. The pellet was
subjected to another wash in TEX buffer. Finall y t he pellet
was resuspended in 100 µl of TEX buffer and was lysed
by heating in a dry bath at 95˚C for 15min [18]. The lys-
ate was used as the DNA template for PCR. This lysate
could be stored at 2˚C - 8˚C for 2 - 3 months and at
–20˚C for > 1 year.
2.2. Polymerase Chain Reaction
All the primer sequences listed Table 1 were procured
from Eurofins Genomics India Pvt Ltd. Multiplex-PCR
mixture for mecA, femA and 16S rRNA consisted of the
following; 1 × PCR mixture (Fermentas Life Sciences/
HiMedia) containing 1.5 mM MgCl2, 50 pM of each
primer, 200 µM of each dNTP along with 1 U of Taq
(HiMedia) and 5 µL of DNA template in 20 µL final
volume. PCR reactions were initiated with a denaturation
at 94˚C for 5 mins, 40 cycles of denaturation (94˚C, 30 s),
annealing (55˚C, 40 s) and primer extension (72˚C, 50 s),
with a final extension at 72˚C for 10mins.
A femA monoplex PCR was performed under the re-
ported conditions [14,19] but with 10 µL of the DNA
template. Monoplex PCR was also optimized for the
identification of the nuc gene in these isolates. The PCR
reaction mixture contained in 20 µL final volume 50 pM
of nuc pr imers and 5 µl of DNA lysate in addition to 200
µM of dNTPs, and 1 unit of Taq DNA polymerase.
The PCR reactions with femA flanking primers were
performed in a volume of 20 µL containing 10 µL of DNA
lysate, containing 1.7 mM MgCl2, 100 pM of primers,
0.2 mM dNTPs along with 2.5 units of Taq DNA poly-
merase. A 30-cycle amplification followed an initial de-
naturation at 94˚C for 5 min, with d enaturation (94˚C, 30
s), annealing (45˚C, 1 min) and primer extension (72˚C,
45 s), and final extension at 72˚C for 10 mins.
3. Results
3.1. 16s rRNA, femA and mecA Multiplex PCR
Of the 54 isolates which were included in the study, 21
(38%) were isolated from blood, 13 (24%) from pus, 9
(16.6%) from wound swabs and the rest from other
sources such as sputum, tissue, endo-tracheal secretions
and others. These isolates were identified as MRSA (26
isolates), MSSA (26 isolates) and CoNS (2 isolates).
Multiplex-PCR was performed targeting 16S rRNA for
the Staphylococci genus, femA for S. aureus and mecA
for methicillin resistance. All the isolates tested positive
for the genus specific 16S rRNA 886 bp product. The
293 bp mecA was detected in all 26 (100%) MRSA iso-
lates, 16 (61.5%) MSSA isolates and 1 CoNS isolate.

Heterogeneit y in femA in the Indian Isolates of Staphylococcus aureus Limits Its Usefulness
as a Species Specific Marker
84
Table 1. Information related to the PCRs performed in this study.
Name of
the Primers Sequence 5’-3’ Product Size bp Reference
mecA-F ACG AGT AGA TGC TCA ATA TAA
mecA-R CTT AGT TCT TTA GAG ATT GA 293 bp
femA-F CGA TCC ATA TTT ACC ATA TCA
femA-R ATC ACG CTC TTC GTT TAG TT 450 bp
[19]
16s-1 GTG CCA GCA GCC GCG GTA A
16s-2 AGA CCC GGG AAC GTA TTC AC 886 bp [20]
nuc1 GCG ATT GAT GGT GAT ACG GTT
nuc2 AGC CAA GCC TTG ACG AAC TAA AGC 270 bp [15]
fem-A1 AGA CAA ATA GGA GTA ATG AT
fem-A2 AAA TCT AAC ACT GAG TAA TGA T 509 bp [14]
FSF-femA AAC GAG AGA CAA ATA GGA GTA ATG A
FSR-femA TCA TGT TTT GAT AAT TCC CTT CC 1312 bp
mecA-F: mecA-Forward Primer, mecA-R: mecA-Reverse, femA-F: femA-Forward Primer, femA-R: femA-Reverse Primer, 16s-1: 16S rRNA Forward Primer,
16s-2: 16S rRNA Reverse Primer, nuc1: nuc Forward Primer, nuc2: nuc Reverse Primer, fem-A1: femA Forward Primer, fem-A2: femA Reverse Primer,
FSFfemA: Flanking Sequence femA forward Primer, FSRfemA: Flanking Sequence femA Forward Primer.
It was interesting to note that only 16 (29.6%) isolates
showed amplification of the 450 bp femA gene (9 MRSA,
7 MSSA) with both the CoNS isolates were negative for
femA.
3.2. Monoplex PCR of the Species-Specific femA
and nuc Genes
As femA failed to show up in the multiplex PCR, femA
monoplex-PCR was done using the primers reported by
Al-Talib et al. [19] and Kobayashi et al. [14]. Both these
sets of primers failed to identify the femA in those iso-
lates, thereby conf irming our results of multiplex PCR.
As femA is generally accepted as a species specific
marker, we wanted to corroborate femA results with an-
other species specific marker, namely the thermostable
nuclease gene, nuc, to confirm S. aureus and compared
the sensitivities of femA and nuc for detecting S. aureus.
Out of the 54 isolates, 29 (53.7%) show ed the 270 bp nuc
that included 52% MRSA and 48% MSSA, while both
the CoNS isolates were negative for nuc amplification.
3.3. Amplification of the Full Length femA
Using Primers from the Flanking Regions
To further investigate the reason for the non -a mpl if ic at io n
of the femA gene in most of the isolates, assuming inter-
nal sequence variations, we designed primers flanking the
femA gene (27 bp upstream and 22 bp downstream to
femA) to amplify the full length femA gene of 1263 bp,
giving a PCR product of 1312 bp. This was done as fol-
lows. 11 femA gene sequences of Staphylococcus aureus
submitted in the GenBank data base were downloaded
and a ClustalW analysis was performed. Primers for femA
gene flanking sequences were designed using the online
tool Primer3 and the expected product size was 1312bp.
The designed primers were blasted against all the 11
femA sequences from the GenBank database and the
primers showed 100% alignment with all the femA se-
quences (Table 2).
13 of the 54 isolates, including 7 MRSA and 6 MSSA
showed amplification of full length femA gene. Twelve
of them, showing the exp ected 1312 bp product were the
femA positive isolates and one was about 700 bp trun-
cated product (Figure 1). All the other femA negative
isolates failed to show any amplification using these
primers (see also Table 3).
4. Discussion
In this study, during our screening for methicillin resis-
tance markers in clinical isolates, we observed that the
femA species specific marker failed to amplify in several
isolates. We present evidence to show that femA cannot
be used as a reliable marker for S. aureus in this geogra-
phical region. We demonstrated this using both multiplex
and monoplex PCR.
There has been a tremendous increase in the number of
MRSA cases in the past few years. Therefore, several
efforts have been made for quicker and early detection of
MRSA. Molecular methods have now become the gold-
standard for rapid detection of MRSA [12,21]. Several
studies have reported the use of mecA as marker for de-
tection of methicillin resistance, fem genes and nuc for

Heterogeneit y in femA in the Indian Isolates of Staphylococcus aureus Limits Its Usefulness
as a Species Specific Marker 85
Table 2. List of femA gene sequences reviewed from the Ge nBank Database .
S.No Strain GenBank accession Gene location
A:
1 MRSA252 NC_002952 1444550 - 144581 2
2 USA300_FRP3757 NC_007793 1397007 - 1398308
3 JKD6008 NC_017341 1427188 - 142845 6
4 COL NC_002951 1420810 - 142211 1
5 Newman NC_009641 1417031 - 1418293
6 ED98 NC_013450 1391969 - 1393231
7 USA300_TCH 1516 NC_010079 1410993 - 1412255
8 TCH60 NC_017342 1939657 - 1940994
9 RF122 NC_007622 1344801 - 1346102
10 MW2 NC_003923 1381193 - 1382455
11 Mu3 NC_009782 1456933 - 1458195
B:
12 JKD6159 NC_017338 1365979 - 1367241
13 N315 NC_002745 1379204 - 1380466
14 Mu50 NC_002758 1455533 - 1456795
15 T0131 NC_017347 1428806 - 1430068
16 04-02981 NC_017340 1413795 - 1415057
17 HO 5096 0412 NC_017763 1357669 - 135893 1
18 TW20 NC_017331 1470704 - 1471966
Lane 1: Low Range Ladder, Lane 2: Multiplex-PCR [16S rRNA (886bp), femA (450 bp), mecA (293bp)], Lane 3:
femA-Al-Talib (450 bp), Lane 4: femA-Kobayashi (509 bp), Lane 5: nuc (270 bp), Lane 6: femA full length (1312 bp),
Lane 7: femA full le ngth (mutated, approx. 700 bp), Lan e 8: Negative Control.
Figure 1. PCR amplification of molecular targets for MRSA and S. aureus.

Heterogeneit y in femA in the Indian Isolates of Staphylococcus aureus Limits Its Usefulness
as a Species Specific Marker
86
Table 3. Summary of results phenotype vs genotype.
Phenotype
MRSA (26)* MSSA (26)* CoNS (2)* Genotype
+ – + – + –
Total
(54)*
+
mecA 26 (100%) 0 (0%) 16 (61.5%) 10 (38.5%) 1 (50%) 1 (50%) 43 (79.6%)
16S rRNA 26 (100%) 0 (0%) 26 (100%) 0 (0%) 2 (100%) 0 (0%) 54 (100%)
femA 9 (34.6%) 17 (65.4%) 7 (26.9%) 19 (73.1%) 0 (0%) 2 (100%) 16 (29.6%)
nuc 15 (57.7%) 11 (42.3%) 14 (53.8%) 12 (46.2%) 0 (0%) 2 (100%) 29 (53.7%)
femA full length 7 (26.9%) 19 (73.1%) 6 (23.1%) 20 (76.9%) 0 (0%) 2 (100%) 13 (24%)
*Total Number of Isolates.
identification of S. aureus species. Good correlation of
phenotype with genotype tests were reported for MRSA
isolates, which harboured mecA and femA genes [19,22].
In the study by Kobayashi et al, mecA was detected in
100% of MRSA, 16.7% of MSSA isolates. 10.6% of
their S. aureus isolates did not amplify femA gene PCR
product [14]. Thermostable nuclease gene nuc was re-
ported to have 100% sensitivity and specificity for the
identification of S. aureus isolates [15,23]. In India, only
a few studies have reported the use of femA and nuc
along with mecA as molecular targets for identification of
S. aureus and characterisation of MRSA [24,25] and
there are no reports which specifically investigated the
predictive value of femA gene for the identification of S.
aureus. Variations (polymorphism) in the genomic se-
quences are not uncommon in S. aureus, even highly
conserved and widely used species specific markers like
coagulase (coa), Staphylococcal Protein A (spa) genes
have shown polymorphisms. Variations in the sequence
of the coa and spa genes and the hyper variable region
adjacent to the mecA gene, have been the basis for the
most widely used forms of PCR typing of MRSA [26-28].
Further, it is also known that sequence variations culmi-
nate in changes in virulence properties of S. aureus which
influence clinical disease manifestations in humans [29].
The mecA PCR in our study showed absolute correla-
tion to MRSA phenotype and we observed a rather high
rate (58.3%) of detection of mecA among MSSA isolates.
An intriguing finding in this study was the non-amplifi-
cation of femA gene in most (70.4%) of the isolates when
screened with two different sets of primers for the femA
gene and confirmed by non-ampli fication of the full length
femA gene with the primers flanking the femA gene.
Though nuc PCR was more sensitive (57.4%) compared
to femA PCR (29.6%) in the detection of S. aureus we
could not achieve 100% sensitivity with nuc PCR also.
The femA PCR and nuc PCR showed good correlation in
15 out of 16 isolates. However, 22 S. aureus isolates were
negative for both femA and nuc genetic markers. This is
suggestive of likely mutations or deletions in the nuc
gene also. However, further investigations are being car-
ried out to understand this observation.
Mutations in the aux illary genes such as the fem, cou ld
explain the absence of any phenotypic expression of re-
sistance in MSSA isolates though these isolates con-
tained mecA gene [30,31]. We propose to examine this
phenomenon in future in several MSSA isolates. All the
MRSA isolates that showed positive femA PCR, had
MICs greater than 256 µg/mL; yet there were 3 isolates
with MICs > 256 µg/mL that showed no femA gene am-
plification. It is known that methicillin resistance could
manifest with or without mecA. Auxillary genes like
femA influence the extent of resistance [14] implying that
there could be other mechanisms that circumvent absence
of femA gene to confer methicillin-resistance.
Assuming that mutations in the femA gene sequence at
the primer annealing sites could have resulted in non-am-
plification of PCR product in some isolates, we attemp ted
to amplify the full length femA gene by designing prim-
ers flanking femA as described previously. Surprisingly,
amplification of the whole gene was seen in only in some
of those isolates which were positive with femA primers.
One MSSA isolate which was negative for femA showed
amplification of a truncated (smaller) (app roximately 7 00
bp instead of 1312 bp) product, sugg esting possible dele-
tions in the femA gene. We are sequencing this smaller
PCR product. Four femA positive isolates did not amplify
the full length PCR product indicating that there are
variations in primer annealing sites flanking the femA
gene. Re-visit to the GenBank database for any new en-
tries of femA gene, we found 7 new sequences of S. aureus
that have recently been submitted in April/May 2012
(Table 2, B) which showed significant variations in their
sequences compared to the 11 femA gene sequences (Ta-
ble 2, A) which were used in the design of the PCR
primers that we evaluated in this study. Of these se-
quences, a strain JKD6159 showed two nucleotide varia-
tions in the sequence flanking the femA gene (4th & 13th
base downstream of femA) which could probably explain
the non-amplification of the full length femA gene se-
quence in our isolates also. Significant polymorphisms in
the recently submitted femA gene sequences suggest the

Heterogeneit y in femA in the Indian Isolates of Staphylococcus aureus Limits Its Usefulness
as a Species Specific Marker 87
femA gene variations are quite common, could be region
specific, may play important role in the expression of
MRSA and in the identification of S. aureus in clinical
isolates. Our study shows that polymorphisms in femA
gene sequences are present in both MRSA and MSSA
isolates in India and requires further investigations such
as sequencing to characterise the genome in general and
femA in particular. We have initiated a detailed investi-
gation of genetic variations in th e femA gene of S. aureus,
its relevance to the drug resistance phenotype and to ex-
amine any role for these genomic variatio ns in molecular
epidemiology of clinical isolates of S. aureus.
5. Acknowledgements
Technical assistance by Mr. T. Naresh Singh of Clinical
Microbiology Laboratory is acknowledged and appreci-
ated.
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