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

Abstract

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.
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.
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.
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.

Full text

Methicillin-resistant Staphylococcus aureus
(MRSA) is a major cause of nosocomial infections
worldwide1,2. All species of staphylococci have been
identied as pathogen in blood stream infections3,
skin and soft tissue infections, post-operative wound
infections and in ocular infections as well4-7.
Application of PCR based - RFLP for species identification of ocular
isolates of methicillin resistant staphylococci (MRS)
J. Malathi, M. Sowmiya, S. Margarita, H.N. Madhavan & K. Lily Therese
L&T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, India
Received April 25, 2008
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 conrmed 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 40C and sub-cultured at every 15 days interval.
Results: Seventy (63.6%) of 110 isolates were identied 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
identied 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
Rajaduraipandi et al8 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 MRSA9. Of them, 1.3 per
Indian J Med Res 130, July 2009, pp 78-84
78

cent 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 occurred9.
Coagulase-negative staphylococci (CNS) are the
most common pathogens causing endophthalmitis7 and
most cases of infectious endophthalmitis occurring
after cataract surgery are due to bacteria entering the
eye at the time of surgery10. Laboratory diagnosis and
susceptibility testing are crucial in treating, controlling,
preventing MRS infections1.
MRS has ability to grow in presence of derivatives
of beta-lactams1-3,11. Methicillin resistance (MR) is
transferred to susceptible strains through horizontal
transfer of mecA gene12. The mecA gene encodes
penicillin binding protein, PBP2a2,13 and it is a useful
molecular marker of putative MR2. Detection of MR
by conventional method is time consuming, inuenced
by antibiotics, culture medium, NaCl 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 quickly1,14.
The femB gene codes for an enzyme important in
cross-linking peptidoglycan in various staphylococci.
The specicity of the femB PCR primers used for
DNA amplication in S. aureus has been demonstrated
previously15.
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 identication 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 elsewhere16. 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 sub-
cultured onto stock agar (Hyer’s and Johnson agar),
which was made in penicillin bottles as stab cultures and
maintained at 40C. The organism were retrieved from
the stock culture by sub culturing onto blood agar and
incubated at 370C for 18-24 h. Also these organisms
were periodically re sub-cultured at 15 days duration.
Conventional method for identication of S. aureus
and methicillin resistance: Staphylococcal isolates
were identied 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 NaCl tolerance. Tube
coagulase production is considered “gold standard” for
identication of S. aureus1.
Detection of MR by disc diffusion method for S. aureus:
Methicillin disc (5μg) (Hi-Media Laboratories Private
Limited, Mumbai) was placed on Muller-Hinton agar
with 5.0 per cent NaCl according to CLSI guidelines and
incubated for 24 h at 35 ± 2ºC17. 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 > 14mm17. 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 µg),
ciprooxacin (5 µg), moxioxacin (5 µg), noroxacin
(10 µg), gentamicin (10 µg), tobramycin (10 µg),
ooxacin (5 µg), and penicillin G (10 units) by Kirby
bauer disc diffusion method17. 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
µg/ml in deionized water. Antibiotic was serially
diluted in Muller-Hinton broth with 2.0 per cent NaCl
to give working concentrations of 64 - 0.125 µg/ml18
and bacterial suspension of 0.5 McFarland turbidity
MALATHI et al: PCR-RFLP FOR DETECTION OF MRS 79

standards (containing approximately 1 to 2 x 108 cfu/
ml) was added to all the tubes and were incubated for
24 h at 35 ± 2ºC18. The MIC breakpoint of oxacillin (1
µg/ml) for S. aureus is > 4.0 - < 2.0 (µg/ml) and for
CNS it is > 0.5 - < 0.25 (µg/ml).
Molecular methods: DNA was extracted using a single
colony from an overnight culture of Staphylococci by
modied guanidine thiocyanate (GTC) method19 with
modication where proteinase K and lysostaphin 1 μg/
μl (Sigma-Aldrich-L0761 - Staphylococcus simulans
(USA) were added for enhancing digestion at the initial
step).
All PCR reagents used for amplication including
primers were procured from Bangalore Genei, Ltd.
Bangalore, India. All PCR amplications 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 al2 were
used. From the extracted DNA, 5 µl was added to
45 µl of PCR mixture consisting of 5 μl buffer (10X
buffer containing 15 mM MgCl2) 200 μM dNTPs, 3
mM MgCl2, 75 picomoles of each femB / 20 picomoles
of mecA primer, 30 μl 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 prole consisted
of initial denaturation at 94°C for 5 min followed by10
cycles with denaturation at 94°C for 30 sec, annealing at
640C for 30 sec, and extension at 72°C for 45 sec and
further 35 cycles consisting of denaturation at 94°C for 45
sec, annealing at 50°C for 45 sec, and extension at 72°C
for 1 min followed by nal extension at 72°C for 10 min.
Analytical sensitivity of uniplex PCR was
determined using serial ten-fold dilutions of
DNA extracts of positive and negative controls.
Specicity was determined with DNA extracts of
standard strains of Mycobacterium tuberculosis
(H37RV), M. xenopi, (ATCC-1432), Haemophilus
inuenzae (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 identication of various species
of MRS
PCR of gap gene - PCR for gap gene was performed
only for 70 methicillin resistant isolates using primers
described20,21. PCR was carried using 5 µl of DNA
extracted added to 45 µl of PCR mixture consisting of
5 μl 10X PCR buffer containing 15 mM Mgcl2, 200 μM
dNTPs, 5 picomoles of forward and reverse primers,
30 μl deionized water of and 1.25U of Taq polymerase.
Thermal prole followed was initial denaturation at
94°C for 2 min, followed by 40 cycles consisting of
denaturation at 94°C for 20 sec, annealing at 55°C for
30 sec, and extension at 72°C for 40 sec and a nal
extension at 72°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 amplied
product was done using the restriction enzyme AluI.
Reaction mixture (30 μl) consisted 17 μl deionized
water, 10 μl PCR product, 3 μl buffer, 0.4 μl restriction
enzyme (10 units/μl, Fermentas Life Science, USA) and
incubated at 37°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 Weller13 except
DNA template taken is 20-30 ng.
Cycle sequencing reaction, consisted of 4 μl of big
dye terminator, 2 μl of DNA, 2 picomoles/μl of forward
reverse primer, 3 μl of deionized water. PCR prole
consisted of denaturation at 96°C for 1 min, followed
by 25 cycles of 96°C for 10 sec, 50°C for 5 sec and
60°C for 4 min and nal extension of 4°C. Products
were puried according to standard protocol, loaded
onto ABI 3100 Genetic Analyzer (Applied Biosystem,
USA) with polymer POP6 and sequenced. Sequences
80 INDIAN J MED RES, JULY 2009

MALATHI et al: PCR-RFLP FOR DETECTION OF MRS 81
were analyzed using BIO EDIT sequence alignment
software22, CHROMAS23 and nally blasted with
NCBI24 to identify the species and DNA homology.
Results
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
identied as S. aureus based on the results of tube
coagulase, mannitol fermentation and growth in 7.5
per cent NaCl 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 µg/ml
by oxacillin, 22 (20%) had 16 µg/ml, 10 (9.1%) each
had 8, 4 and <2 µg/ml. Ten isolates (9.1%) had MIC
<0.5 µg/ml and remaining 40 (36.3%) had MIC <0.125
µg/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 noroxacin, 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 specic 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
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
for S. aureus
No. (%) negative
for S. aureus
Conventional
method
(coagulase test)
PCR for
femB gene
Conventional
method
(coagulase test)
PCR for
femB gene
23
(20.9)
23
(20.9)
87
(78.1)
87
(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
No. (%) negative
for methicillin resistance
Conventional
method
PCR for
mecA gene
Conventional
Method
PCR for
mecA gene
Disc diffusion
Method
Microbroth dilution
method
Disc
diffusion
method
Microbroth dilution
method
70
(63.6)
70
(63.6)
70
(63.6)
40
(36.4)
40
(36.4)
40
(36.4)
¶ = MIC break point of oxacillin (1 µg/ml): For S. aureus = ≥ 4.0 - ≤ 2.0 (µg/ml); For CNS = ≥ 0.5 - ≤ 0.25 (µg/ml)
Table III. Results of antibiotic susceptibility testing showing number of MRS
Species (n) Cefazolin Ciprooxcin Moxioxacin Noroxacin Gentamicin Tobramycin Ooxacin Penicillin
S.aureus (18) 10 11 2 15 16 17 8 18
S. haemolyticus (27) 11 11 3 24 22 24 4 27
S. epidermidis (11) 1 5 2 8 7 7 1 11
S. cohnii subsp.
urealyticum (6)
2 2 0 3 3 3 0 6
S. equorum (6) 1 3 0 4 5 5 3 6
S. hominis (1) 0 0 0 0 1 1 0 1
S. xylosus (1) 0 0 0 0 0 0 0 1
Total (70) 25 32 7 54 54 57 16 70

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 identied 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
conrmed 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).
Discussion
MRSA and MR-CNS are predominant bacterial
pathogens isolated from ocular specimens4-6.
Conjunctivitis is the most commonly reported
manifestation8. 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 al5, S. epidermidis
was identied 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 & Kumar25
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 identication,
Table IV. Comparison of results of RFLP and DNA sequencing on
identification of Staphylococcus spp.
Species identied
by RFLP
Total no.
(%)
DNA sequencing results
% homology and
species identied
by forward primer
% homology
and species
identied by
reverse primer
S.aureus 18 (25.71) 97
S. aureus
98
S. aureus
S. epidermidis 11 (15.71) 96
S. epidermidis
100
S. epidermidis
S. haemolyticus 27 (38.57) 98
S. haemolyticus
99 S.
haemolyticus
S. cohnii subsp.
urealyticum
6 (8.57) 96
S. cohnii subsp.
urealyticum
96
S. cohnii subsp.
Urealyticum
S. equorum 6 (8.57) 99
S. equorum
99
S. equorum
S. xylosus 1 (1.42) 97
S. xylosus
97
S. xylosus
S. hominis 1 (1.42) 97
S. hominis
98
S. hominis
Standard strains:
S.aureus (ATCC 6538) 98
S. aureus
98
S. aureus
S.epidermidis (ATCC 12228) 100
S.epidermidis
100
S.epidermidis
Staphylococcus
saprophyticus
100
S.saprophyticus
100
S.saprophyticus
Fig. 2. Agarose gel electrophoretotogram showing the results
of mecA gene positive for DNA extracted from isolates. Lane 1:
negative control; Lane 2: extraction control; Lanes 3-13: showing
amplied mecA gene in isolates; Lane 14: PC S.aureus MRSA
(ATCC 33591); Lane 15: MW - 100 bp ladder.
Fig. 1. Agarose gel electrophoretotogram showing the results
of femB gene positive for DNA extracted from isolates. Lane 1:
negative control; Lane 2: extraction control; Lanes 3-8 showing
amplied femB gene in isolates; Lanes 9 and 10: show absence of
femB gene in isolate; Lane 11: PC S.aureus MSSA ATCC 6538;
Lane 12: MW - 100 bp ladders.
82 INDIAN J MED RES, JULY 2009

as it is cost-effective compared to application of API
ID32 system used by others for similar type of study5.
Present study showed that 24 h incubation period
was sufcient 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 <0.1 µg of oxacillin/ml (instead of <0.5
µg of oxacillin/ml) for CNS17,18 permitted detection of
10 more CNS isolates with mecA-associated resistance.
Kohner et al26 also found more of CNS resistant strains
with breakpoints of <0.1µg/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 specic for
staphylococcal species.
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Fig. 3. Agarose gel electrophoresis of fragments produced by AluI
digestion of 933-bp PCR amplication products from Staphylococcus
species. Lane 1: negative control; Lane 2: Undigested product (933
bp); Lane 3: S.saprophyticus lab isolate; Lane 4: S. haemolyticus;
Lane 5: S. xylosus; Lane 6: S. hominis; Lane 7: S. cohnii subsp.
urealyticum; Lane 8: S. equorum; Lane 9: S. epidermidis; Lane 10: S.
aureus; Lane 11: S. aureus (ATCC 6538); Lane 12: S. epidermidis
(ATCC 12228); Lane 13: 100 bp ladder.
MALATHI et al: PCR-RFLP FOR DETECTION OF MRS 83

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