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Application of PCR based--RFLP for species identification of ocular isolates of methicillin resistant staphylococci (MRS).

Background & objectives: Early detection of methicillin resistant staphylococci (MRS) from clinical specimens enables institution of appropriate antimicrobial therapy. Limited information is available on speciation of MRS. This study was undertaken to compare results of conventional and molecular methods in detection of methicillin resistance (MR) and application of PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing for speciation of ocular isolates of MRS.

Methods: A total of 110 consecutive ocular staphylococcal isolates were screened for MR. MRS was speciated by PCR-RFLP of gap gene and results were confirmed by DNA sequencing. All isolates were processed within 48 h of isolation. A single colony of bacterium, stocked as stab cultures in Hyer's and Johnson agar, was stored at 4[degrees]C and sub-cultured at every 15 days interval.

Results: Seventy (63.6%) of 110 isolates were identified as MRS and 40 (36.4%) were MSS by conventional and molecular method (100% correlation). Of the 70 MRS, 18 (25.7%) were Staphylococcus aureus, remaining 52 (74.3%) were CNS by conventional and molecular method (100% correlation). PCR-RFLP of gap gene identified 18 (25.71%) MRS as S. aureus, 11 (15.71%) S. epidermidis, 27 (38.57%) S. haemolyticus, 6 (8.57%) S. cohnii subsp. urealyticum, 6 (8.57%) S. equorum, 1 (1.42%) S. xylosus and 1 (1.42%) S. hominis.

Interpretation & conclusions: Overall rate of isolation MRS was 63.6 per cent and were predominantly isolated from conjunctival swab (23.6%) and donor corneal scleral rim (23.6%) of non hospitalized patients indicating their community origin. Detection of MR by mecA gene was easier and less time consuming compared to conventional methods. Speciation of MRS was possible by gap gene PCR--RFLP and the predominant MRS in our study was S. haemolyticus.

Key words Coagulase negative staphylococci--methicillin resistance--methicillin resistant staphylococci--methicillin sensitive staphylococci--restriction fragment length polymorphism


Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections worldwide (1,2). All species of staphylococci have been identified as pathogen in blood stream infections (3), skin and soft tissue infections, post-operative wound infections and in ocular infections as well (4-7).

Rajaduraipandi et al (8) found 31.1 and 37.9 per cent of S. aureus methicillin resistant among clinical and carrier samples respectively. In another study 30 per cent of patients were considered to have acquired MRSA via nosocomial transmission and 70 per cent to had community acquired MRSA (9). Of them, 1.3 percent had ophthalmic MRSA involvement. The most common manifestation of ophthalmic MRSA infection was preseptal cellulitis and/or lid abscess followed by conjunctivitis, but sight-threatening infections, including corneal ulcers, endophthalmitis, orbital cellulitis, and blebitis, also occurred (9).

Coagulase-negative staphylococci (CNS) are the most common pathogens causing endophthalmitis (7) and most cases of infectious endophthalmitis occurring after cataract surgery are due to bacteria entering the eye at the time of surgery (10). Laboratory diagnosis and susceptibility testing are crucial in treating, controlling, preventing MRS infections (1).

MRS has ability to grow in presence of derivatives of beta-lactams (1-3,11). Methicillin resistance (MR) is transferred to susceptible strains through horizontal transfer of mecA gene (12). The mecA gene encodes penicillin binding protein, PBP2a (2,13) and it is a useful molecular marker of putative MW. Detection of MR by conventional method is time consuming, influenced by antibiotics, culture medium, NaC1 concentration, temperature and time of incubation. PCR-based methods for detection of MR by mecA gene, is considered 'gold standard' and the results can be obtained quickly (1,14).

The femB gene codes for an enzyme important in cross-linking peptidoglycan in various staphylococci. The specificity of the femB PCR primers used for DNA amplification in S. aureus has been demonstrated previously (15).

There are only a few reports available on ocular MRS in literature. This study was undertaken to determine the rate of isolation of MRS among ocular isolates by conventional and molecular methods and to standardize, apply PCR-RFLP and DNA sequencing techniques for identification and speciation of MRS and to compare the results of conventional and molecular methods in the detection of MR.

Material & Methods

Bacterial strains: One hundred and ten consecutive staphylococcal isolates recovered from ocular clinical specimens (conjunctival swab--46, donor corneal rim--36, corneal scraping--9, anterior chamber tap--1, corneal button--6, vitreous aspirate--1, others--11) received at L & T Microbiology Laboratory, Sankara Nethralaya, a tertiary eye care centre at Chennai, India, during November 2005--August 2006, were included in this study. The study protocol was approved by the institutional ethics sub-committee. Clinical specimens were processed as described elsewhere (16). Gram-positive cocci in more than one medium were included.

A single colony of the bacterial isolates after various analysis was picked up from 24 h old plate and subcultured onto stock agar (Hyer's and Johnson agar), which was made in penicillin bottles as stab cultures and maintained at 4[degrees]C. The organism were retrieved from the stock culture by sub culturing onto blood agar and incubated at 37[degrees]C for 18-24 h. Also these organisms were periodically re sub-cultured at 15 days duration.

Conventional method for identification of S. aureus and methicillin resistance: Staphylococcal isolates were identified by Grams staining, catalase production, haemolysis on blood agar, oxidative-fermentative test, production of bound and free coagulase, mannitol fermentation and 7.5 per cent NaC1 tolerance. Tube coagulase production is considered "gold standard" for identification of S. aureus (1).

Detection of MR by disc diffusion method for S. aureus: Methicillin disc (5[micro]g) (Hi-Media Laboratories Private Limited, Mumbai) was placed on Muller-Hinton agar with 5.0 per cent NaC1 according to CLSI guidelines and incubated for 24 h at 35 [+ or -] 2[degrees]C (17). MSSA (ATCC 6538) and MRSA (ATCC 33591) were included as controls. Interpretative criteria for disc diffusion method for S. aureus: resistant < 9mm, intermediate resistance 10-13 mm and sensitive [greater than or equal to] 14mm (17). In order to distinguish strains exhibiting "Intermediate resistant" from that of "heterogenous strains" the sensitivity plates with strains exhibiting resistance were incubated for an additional 24 h. At the end of 48 h of incubation, heterogenous strain turned sensitive whereas intermediate resistant strains remained resistant.

All coagulase negative staphylococci were tested for antibiotic susceptibility against cefazolin (30 [micro]g), ciprofloxacin (5 [micro]g), moxifloxacin (5 [micro]g), norfloxacin (10 [micro]g), gentamicin (10 [micro]g), tobramycin (10 [micro]g), ofloxacin (5 [micro]g), and penicillin G (10 units) by Kirby bauer disc diffusion method (17). All the antibiotics were obtained from Hi-Media Laboratories Private Limited, Mumbai.

Detection of MIC by microbroth dilution method for oxacillin: Oxacillin, (Sigma-Aldrich Company, USA) stock solution was prepared at concentrations of 1280 [micro]g/ml in deionized water. Antibiotic was serially diluted in Muller-Hinton broth with 2.0 per cent NaC1 to give working concentrations of 64 - 0.125 [micro]g/ml (18) and bacterial suspension of 0.5 McFarland turbidity standards (containing approximately 1 to 2 x [10.sup.8] cfu/ ml) was added to all the tubes and were incubated for 24 h at 35 [+ or -] 2[degrees] (18). The MIC breakpoint of oxacillin (1 [micro]g/ml) for S. aureus is [greater than or equal to] 4.0 [less than or equal to] 2.0 ([micro]g/ml) and for CNS it is > 0.5 [less than or equal to] 0.25 ([micro]g/ml).

Molecular methods: DNA was extracted using a single colony from an overnight culture of Staphylococci by modified guanidine thiocyanate (GTC) method (19) with modification where proteinase K and lysostaphin 1 [micro]g/ [micro]l (Sigma-Aldrich-L0761--Staphylococcus simulans (USA) were added for enhancing digestion at the initial step).

All PCR reagents used for amplification including primers were procured from Bangalore Genei, Ltd. Bangalore, India. All PCR amplifications were carried out using PCR thermal cycler Perkin Elmer Model 2700 (Applied Biosystems, Massachusetts, USA).

Uniplex PCR for the detection of femB and mecA gene: PCR conditions were optimized according to laboratory conditions. Both mecA and femB PCR primer sequences described by Perez-Roth et al (2) were used. From the extracted DNA, 5 [micro]l was added to 45 [micro]l of PCR mixture consisting of 5 [micro]l buffer (10X buffer containing 15 mM Mg[Cl.sub.2]) 200 [micro]M dNTPs, 3 mM Mg[Cl.sub.2], 75 picomoles of each femB / 20 picomoles of mecA primer, 30 [micro]l deionized water and 1.25U Taq polymerase.

Both uniplex PCRs were carried out with negative and positive control containing S. aureus-MSS (ATCC 6538) and MRSA (ATCC 33591). PCR profile consisted of initial denaturation at 94[degrees]C for 5 min followed by 10 cycles with denaturation at 94[degrees]C for 30 sec, annealing at 64[degrees]C for 30 sec, and extension at 72[degrees]C for 45 sec and further 35 cycles consisting of denaturation at 94[degrees]C for 45 sec, annealing at 50[degrees]C for 45 sec, and extension at 72[degrees]C for 1 min followed by final extension at 72[degrees]C for 10 min.

Analytical sensitivity of uniplex PCR was determined using serial ten-fold dilutions of DNA extracts of positive and negative controls. Specificity was determined with DNA extracts of standard strains of Mycobacterium tuberculosis (H37RV), M. xenopi, (ATCC-1432), Haemophilus influenzae (ATCC-10211), S. aureus (ATCC-6538), S. epidermidis (ATCC-10211), S. pyogenes (ATCC-12384), Streptococcus pneumoniae (ATCC-6301), Acinetobacter calcoaceticus (ATCC-9956), Enterococcus faecalis (ATCC-49149), Escherichia coli (ATCC-4157), Pseudomonas aeruginosa (ATCC 9742), Propionobacterium acne (ATCC-11828) and laboratory isolates of Corynebacterium xerosis (conjunctival swab), Nocardia asteroides (canalicular pus), Actinomyces spp. (canalicular pus), Bacillus cereus (eviscerated material).

Molecular methods for identification of various species of MRS

PCR of gap gene--PCR for gap gene was performed only for 70 methicillin resistant isolates using primers described (20,21). PCR was carried using 5 [micro]l of DNA extracted added to 45 [micro]l of PCR mixture consisting of 5 [micro]l 10X PCR buffer containing 15 mM Mg[cl.sub.2], 200 [micro]M dNTPs, 5 picomoles of forward and reverse primers, 30 [micro]l deionized water of and 1.25U of Taq polymerase. Thermal profile followed was initial denaturation at 94[degrees]C for 2 min, followed by 40 cycles consisting of denaturation at 94[degrees]C for 20 sec, annealing at 55[degrees]C for 30 sec, and extension at 72[degrees]C for 40 sec and a final extension at 72[degrees]C for 5 min.

All PCR products were loaded in 2 per cent agarose gel with ethidium bromide, (Hi-Media Laboratories Private Limited, Mumbai) 50 ng/ml and results were documented in gel documentation system (Vilber Lourmat--France).

RFLP of gap gene--RFLP of the gap gene amplified product was done using the restriction enzyme AluI. Reaction mixture (30 [micro]l) consisted 17 [micro]l deionized water, 10 [micro]l PCR product, 3 [micro]l buffer, 0.4 [micro]l restriction enzyme (10 units//[micro]l, Fermentas Life Science, USA) and incubated at 37[degrees]C water bath for 2 h followed by snap freezing. RFLP products were analyzed using 4 per cent gel electrophoretogram. Staphylococcal isolates with identical RFLP pattern were grouped together and one isolates from each group and three reference strains [S. aureus (ATCC 6538), S. epidermidis (ATCC 12228) and S. saprophyticus--lab isolate] were sequenced using forward, reverse primers of gap gene following the protocol described by Weller (13) except DNA template taken is 20-30 ng.

Cycle sequencing reaction, consisted of 4 [micro]l of big dye terminator, 2 [micro]l of DNA, 2 picomoles/[micro]l of forward reverse primer, 3 [micro]l of deionized water. PCR profile consisted of denaturation at 96[degrees]C for 1 min, followed by 25 cycles of 96[degrees]C for 10 sec, 50[degrees]C for 5 see and 60[degrees]C for 4 min and final extension of 4[degrees]C. Products were purified according to standard protocol, loaded onto ABI 3100 Genetic Analyzer (Applied Biosystem, USA) with polymer POP6 and sequenced. Sequences were analyzed using BIO EDIT sequence alignment software (22), CHROMAS (23) and finally blasted with NCBI (24) to identify the species and DNA homology.


Of 110 staphylococci isolated from varied ocular specimens, maximum number of MRS isolates was from conjunctival swab and donor corneal rim.

All the isolates were Gram positive and catalase positive, utilized glucose. Of the 110 isolates, 23 were identified as S. aureus based on the results of tube coagulase, mannitol fermentation and growth in 7.5 per cent NaC1 and the remaining 87 were CNS.

Among 23 S. aureus, 18 (78.2%) were MRS with inhibition zone of <6 mm disc diffusion method for methicillin and 5 were (21.73%) methicillin sensitive with inhibition zone of > 14mm. Three S. aureus isolates exhibited intermediate resistance at 24 h incubation, became resistant without zone of inhibition, following an additional 24 h of incubation.

By microbroth dilution method, of the 110 staphylococcal isolates, 8 (7.2%) had MIC of 32 [micro]g/ml by oxacillin, 22 (20%) had 16 [micro]g/ml, 10 (9.1%) each had 8, 4 and <2 [micro]g/ml. Ten isolates (9.1%) had MIC <0.5 [micro]g/ml and remaining 40 (36.3%) had MIC <0.125 [micro]g/ml (MSS).

Tables I and II give comparative results of conventional and molecular methods in detection of S. aureus and methicillin resistance.

Maximum number of resistance was observed for tobramycin, followed by norfloxacin, gentamicin. In our study 90 per cent of isolates showed resistance to more than two antibiotics (Table III).

Sensitivity of uniplex PCR for femB gene was 1.3 ng DNA of MSSA-ATCC 6538 and for mecA gene it was 0.77 ng DNA of MRSA-ATCC 33591. Both femB and mecA gene were specific for detection of S. aureus and detection of MR.

Of the 110 staphylococcoal, isolates 18 (16.4%) were positive both for mecA and femB gene (MRSA), 52 (47.2%) were mecA PCR positive and femB PCR negative (MRCNS), 5 (4.5%) others were positive for femB and negative for mecA (MSSA) and remaining 35 (31.8%) were femB and mecA PCR negative (MSCNS). (Figs 1, 2).



Comparing the results of conventional method with molecular method among 110 staphylococci, 18 were tube coagulase, femB and mecA positive (MRSA), 52 were tube coagulase and femB negative and mecA positive (MRCNS). All MRS isolates positive for mecA were MR by disc diffusion, and microbroth dilution methods. Five were tube coagulase and femB positive and mecA negative (MSSA) and 35 were tube coagulase, femB and mecA negative (MSCNS).

Seventy MRS were identified up to species level using PCR-RFLP of gap gene, 18 (25.71%) as S. aureus, 11 (15.71%) as S. epidermidis, 27 (38.57%) S. haemolyticus, 6 (8.57%) S. cohnii subsp, urealyticum, 6 (8.57%) S. equorum; 1 (1.42%) S. xylosus; and 1 (1.42%) S. hominis (Fig. 3). Results of RFLP were confirmed by DNA sequencing and results of forward / reverse primers had higher percentage in blast search. There was 100 per cent correlation between RFLP pattern and sequencing results when blast search was carried out (Table IV).


MRSA and MR-CNS are predominant bacterial pathogens isolated from ocular specimens (4-6). Conjunctivitis is the most commonly reported manifestation (8). These conjunctival swabs were taken from outpatients, showing that MRS was not hospital acquired but was of community origin.

In our study, S. haemolyticus was the predominant MRCNS isolated followed by S. epidermidis. In a similar study conducted by Pinna et al (5), S. epidermidis was identified as a predominant CNS by application of API ID32 system. In many studies, S. epidermidis was predominantly isolated from various clinical specimen followed by S. haemolyticus. Chaudhury & Kumar (25) also reported S. haemolyticus as predominant strain.

Result of PCR-based RFLP on gap gene was 100 per cent in concordance with DNA sequencing results. Maximum intrasequence variation was observed among gap gene of Staphylococci spp., therefore this region could be used for staphylococci species identification, as it is cost-effective compared to application of API ID32 system used by others for similar type of study (5).


Present study showed that 24 h incubation period was sufficient for detecting MRS, but for ruling out heterogeneous resistance, additional 24 h of incubation was needed.

All MRS isolates in our study were positive for mecA gene and all MSS isolates were negative for mecA gene. In this study we demonstrated that breakpoint of [less than or equal to] 0.1 [micro]g of oxacillin/ml (instead of <0.5 [micro]g of oxacillin/ml) for CNS (17,18) permitted detection of 10 more CNS isolates with mecA-associated resistance. Kohner et al (26) also found more of CNS resistant strains with breakpoints of [less than or equal to] 0.1 [micro]g/ml of oxacillin.

In conclusions, isolation rate of MRS was 63.6 per cent among our isolates and MR-CNS constituted a larger portion of MRS. S. haemolyticus, followed by S. epidermidis were predominant CNS isolated. PCR assay was superior in identifying intermediate and heterogeneous MR in shorter duration of time. PCR-RFLP of gap gene was found to be specific for staphylococcal species.

Received April 25, 2008


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Reprint requests: Dr J. Malathi, Reader in Microbiology, L&T Microbiology Research Centre, Vision Research Foundation Sankara Nethralaya, 18 College Road, Chennai 600 006, India e-mail:

J. Malathi, M. Sowmiya, S. Margarita, H.N. Madhavan, K. Lily Therese

L & T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, India
Table I. Comparative results of conventional method with PCR
performed on ocular isolates of Staphylococcus spp. (n=110) for
the identification of S. aureus

 No. (%) positive No. (%)negative
 for S. aureus for S. aureus
 Conventional PCR for Conventional PCR for
 method femB gene method femB gene
(coagulase test) (coagulase test)

 23 23 87 87
 (20.9) (20.9) (78.1) (78.1)

Table II. Comparative results of the conventional method with the
microbroth dilution performed on ocular isolates of Staphylococcus
spp. (n-110) for the detection of methicillin resistance

 No. (%)positive
 for methicillin resistance

 Conventional PCR for
 method mecA gene

 Disc Microbroth
 diffusion dilution
 Method method

 70 70 70
 (63.6) (63.6) (63.6)

 No. (%) negative
 for methicillin resistance

 Conventional PCR for
 method mecA gene

 Disc Microbroth
 diffusion dilution
 method method

 40 40 40
 (36.4 (36.4) (36.4)

([paragraph]) = MIC break point of oxacillin (1 [micro]g/ml): For S.
aureus = [greater than or equal to] 4.0 - [less than or equal to] 2.0
([micro]g/ml); For CNS = [greater than or equal to] 0.5 - [less than
or equal to] 0.25 ([micro]g/ml)

Table III. Results of antibiotic susceptibility testing showing number
of MRS

Species (n) Cefazolin Ciprofloxcin Moxifloxacin

Saureus (18) 10 11 2
S. haemolyticus (27) 11 11 3
S. epidermidis (11) 1 5 2
S. cohnii subsp. 2 2 0
urealyticum (6)
S. equorum (6) 1 3 0
S. hominis (1) 0 0 0
S xylosus (1) 0 0 0
Total(70) 25 32 7

Species (n) Norlioxacin Gentamicin Tobramycin

Saureus (18) 15 16 17
S. haemolyticus (27) 24 22 24
S. epidermidis (11) 8 7 7
S. cohnii subsp. 3 3 3
urealyticum (6)
S. equorum (6) 4 5 5
S. hominis (1) 0 1 1
S xylosus (1) 0 0 0
Total(70) 54 54 57

Species (n) Ofloxacin Penicillin

Saureus (18) 8 18
S. haemolyticus (27) 4 27
S. epidermidis (11) 1 11
S. cohnii subsp. 0 6
urealyticum (6)
S. equorum (6) 3 6
S. hominis (1) 0 1
S xylosus (1) 0 1
Total(70) 16 70

Table IV. Comparison of results of RFLP and DNA sequencing on
identification of Staphylococcus spp.

Species Total no. DNA sequencing results
identified (%)
by RFLP homology and % homology
 species identified and species
 by forward primer identified by
 reverse Inrimer

S.aureus 18 (25.71) 97 98
 S. aureus S. aureus
S. epidermidis 11 (15.71) 96 100
 S. epidermidis S. epidermidis
S. haemolyticus 27 (38.57) 98 99 S.
 S. haemolyticus haemolyticus
S. cohnii subsp. 6 (8.57) 96 96
urealyticum S. cohnii subsp. S. cohnii subsp.
 urealyticum Urealyticum
S. equorum 6 (8.57) 99 99
 S. equorum S. equorum
S. xylosus 1 (1.42) 97 97
 S. xylosus S xylosus
S. hominis 1 (1.42) 97 98
 S. hominis S. hominis

Standard strains:
Saureus (ATCC 6538) 98 98
 S. aureus S. aureus
S.epidermidis (ATCC 12228) 100 100
 Sepidermidis Sepidermidis
Staphylococcus 100 100
saprophyticus S.saprophyticus S.saprophyticus
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Title Annotation:polymerase chain reaction; restriction fragment length polymorphism
Author:Malathi, J.; Sowmiya, M.; Margarita, S.; Madhavan, H.N.; Therese, K. Lily
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Jul 1, 2009
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