Heterogeneity in <i>femA</i> in the Indian Isolates of <i>Staphylococcus aureus</i> Limits Its Usefulness as a Species Specific Marker

doi:10.4236/aid.2012.23013

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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.

Email: *[email protected]

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

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

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

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

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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