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


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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.
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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
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
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,
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
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
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
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
were analyzed using BIO EDIT sequence alignment
software22, CHROMAS23 and nally blasted with
NCBI24 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
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
(coagulase test)
PCR for
femB gene
(coagulase test)
PCR for
femB gene
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
PCR for
mecA gene
PCR for
mecA gene
Disc diffusion
Microbroth dilution
Microbroth dilution
¶ = 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).
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
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
S. aureus
S. epidermidis 11 (15.71) 96
S. epidermidis
S. epidermidis
S. haemolyticus 27 (38.57) 98
S. haemolyticus
99 S.
S. cohnii subsp.
6 (8.57) 96
S. cohnii subsp.
S. cohnii subsp.
S. equorum 6 (8.57) 99
S. equorum
S. equorum
S. xylosus 1 (1.42) 97
S. xylosus
S. xylosus
S. hominis 1 (1.42) 97
S. hominis
S. hominis
Standard strains:
S.aureus (ATCC 6538) 98
S. aureus
S. aureus
S.epidermidis (ATCC 12228) 100
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.
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
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Sankara Nethralaya, 18 College Road, Chennai 600 006, India
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reference to S. haemolyticus. Indian J Med Microbiol 2007;
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26. Kohner P, Uhl J, Kolbert C, Persing D, Cockerill F. Comparison
of susceptibility testing methods with mecA gene analysis
for determining oxacillin (methicillin) resistance in clinical
isolates of Staphylococcus aureus and coagulase-negative
Staphylococcus spp. J Clin Microbiol 1999; 37 : 2952-61.
... However, improvements in diagnosis and identification have revealed that CoNS are an important cause of infected corneal ulcers. Among CoNS, S. haemolyticus is the second most prevalent species causing eye infections [25,26]. Makki et al. reported that 36% of the ocular infections were caused by S. haemolyticus [27]. ...
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Staphylococcus haemolyticus (S. haemolyticus) constitutes the main part of the human skin microbiota. It is widespread in hospitals and among medical staff, resulting in being an emerging microbe causing nosocomial infections. S. haemolyticus, especially strains that cause nosocomial infections, are more resistant to antibiotics than other coagulase-negative Staphylococci. There is clear evidence that the resistance genes can be acquired by other Staphylococcus species through S. haemolyticus. Severe infections are recorded with S. haemolyticus such as meningitis, endocarditis, prosthetic joint infections, bacteremia, septicemia, peritonitis, and otitis, especially in immunocompromised patients. In addition, S. haemolyticus species were detected in dogs, breed kennels, and food animals. The main feature of pathogenic S. haemolyticus isolates is the formation of a biofilm which is involved in catheter-associated infections and other nosocomial infections. Besides the biofilm formation, S. haemolyticus secretes other factors for bacterial adherence and invasion such as enterotoxins, hemolysins, and fibronectin-binding proteins. In this review, we give updates on the clinical infections associated with S. haemolyticus, highlighting the antibiotic resistance patterns of these isolates, and the virulence factors associated with the disease development.
... Detection of MRSA by conventional method is time consuming, influenced by antibiotics, culture medium, NaCl concentration, temperature and time of incubation. There are many published assays for the detection of S. aureus and MRSA by PCR (Jaffe, et al., 2000, lem, et al., 2001, Grisold, et al, 2002, Jonas, et al., 2002, Louie, et al., 2002and Malathi, et al., 2009. Rapid diagnostic tests have the potential to make efforts even more effective. ...
Experiment Findings
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Methicillin-resistant Staphylococcus aureus (MRSA) and sensitive Staphylococcus aureus (SSA) are responsible for a high proportion of nosocomial infections, which makes difficulty in treatment. MRSA infections are responsible for increased mortality rates, longer lengths of hospital stay, and higher rates of treatment failure compared to SSA infections. Detection of MRSA by conventional method is time consuming, influenced by culture medium, concentration of NaCl, temperature, time of incubation and antibiotics. Various PCR methods had been applied for the rapid detection and identification of Staphylococcus species. The dnaJ gene sequence is potentially useful for the identification of genetically related Staphylococcus species and subspecies. While, with other bacterium PCR-restriction analysis is preferred, as a simple and cost-effective method that does not involve radioisotopes. For that reason in this study, we established and evaluated a rapid new protocol of identifying clinically relevant MRSA species by PCR-restriction analysis of the dnaJ gene. SSA and MRSA strains were isolated during a one year period from patients with bacterimia. Identification of S. aureus was performed by standard laboratory methods. Resistance to methicillin was detected by disc diffusion susceptibility test. DNA extraction was performed for both clinical blood samples as well as from isolated SSA and MRSA. Primers were designed to amplify specific dnaJ gene target and confirming the presence of S. aureus. MRSA was speciated by PCR-restriction analysis of dnaJ gene using XapI restriction enzyme. Our results showed two distinguished patterns of PCR-restriction analysis for SSA and MRSA. Only SSA is known to have a XapI restriction sites. Using our protocol, we were able to demonstrate the existence of staphylococcus and to identify their methicillin resistance. Therefore, we suggest that it would be very useful to apply PCR amplification restriction analysis to dnaJ gene directly to clinical specimens early in the diagnostic process. This would save several days that are required for conventional culture. Thus, this established protocol is suggested as a simple and useful method for the rapid detection and simultaneous identification of MRSA in primary clinical specimens or for the identification of culture isolates. This rapid detection would allow clinicians initially to avoid potentially inappropriate treatment options. [Zeinab H. Helal, Fatma Alzahraa M. Gomaa and Sahar M. R. Radwan. New Rapid Method for Differentiation of MRSA and SSA by PCR Restriction Analysis of 920 bp of dnaJ gene. J Am Sci 2012;8(12):832-837]. (ISSN: 1545-1003). 112
... Comparative analyses such as latex agglutination test, tube coagulase test, and coa and nuc gene presence were examined to choose the gold standard method for identification of S. aureus. Tube coagulase test has been used for differentiation of S.aureus in most of the studies (Malathi et al., 2009;Akineden et al., 2011). In one of these studies; latex agglutination test, Slidex Staph plus test and tube coagulase test were compared. ...
... For these reasons, methods based on molecular Elshimy et al. 643 techniques have been developed to stop the spread of MRSA (Giammarinaro et al., 2005). Early detection of MRSA from clinical specimens enables appropriate antimicrobial therapy with an extensive use of antibiotics over the last 50 years which has led to the emergence of bacterial resistance and the dissemination of resistance genes among pathogenic organisms (Malathi et al., 2009;Méndez et al., 2000). ...
... All isolates were identified by catalase production, haemolysis on blood agar, oxidative-fermentative test, and production of bound and free coagulase, manitol fermentation and 7.5 percent NaCl tolerance and heat labile DNase. Tube coagulase production is considered "gold standard" for identification of S.aureus (6). Antimicrobial Susceptibility Test: Disk diffusion test of Penicillin (10 U), vancomycin (30μg), Ampicillin (10µg), Gentamicin (10μg), Erythromycin (15μg), Clindamycin (2μg), Amikacin (30µg), ciprofloxacin (5μg), Tetracycline (30 µg), Co-trimoxazole (25µg) (Mast, Merseyside, United Kingdom), was carried out using Kirby-Bauer Method according to CLSI guidelines 2011. ...
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Introduction: In this study, using the phenotypic and genotypic methods, oxacillin susceptibility in Staphylococcus aureus (S. aureus) strains isolated from patients at two government hospitals in Ilam, Iran was tested. Materials and methods: Out of 200 S. aureus isolates from different human clinical specimens consisting of blood (31%), wound (20%), urine (21%), catheters (7%), sputum (12%), others (9%) were collected. The methicillin resistant S. aureus isolates were investigated using disk diffusion methods and oxacillin (1μg) and cefoxitin (30μg), on MuellerHinton agar were used, and MecA and vanA genes were detected by PCR. In addition, the isolates were tested for their antibiogram profiles. Results: Among 200 S. aureus strains included in this study, 35.96% were MRSA. The percentage of resistance by disk diffusion method was as below: penicillin 85.96%, vancomycin 0%, ampicillin 87.71%, gentamicin 48.25% erythromycin 54.25%, clindamycin 32.45%, amikacin 21.05%, ciprofloxacin 42.10%, tetracycline 51.75% and co-trimoxazole 42.10%. Phenotyping method by disk diffusion method using oxacillin and cefoxitin for detecting of MRSA showed sensitivity and specificity of about 33.33% and 35.96%, respectively. Presence of MecA and vanA genes in MRSA isolates by PCR were 35.96% and 0%, respectively. The oxacillin and cefoxitin disk diffusion methods showed 92.68% and 100% sensitivity, respectively, and 98.8% specificity. Conclusion: Our finding showed that, the cefoxitin disk diffusion method is better in compared to the oxacillin disk diffusion similar to results from detecting of MecA gene in PCR as a golden test.
... Various biochemical tests including catalase test, oxidative-fermentative test, mannitol fermentation, 7.50% NaCl tolerance, and production of bound and free coagulase, using the sheep plasma, were performed for identification of staphylococcal isolates. 20 Antibiotic susceptibility. Antibiotic susceptibility of staphylococcal isolates was determined by disk diffusion method, 21 on Muller-Hinton agar (Merck). ...
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This study was conducted to investigate the prevalence of subclinical mastitis caused by Staphylococcus spp. in ewes in West-Azerbaijan province of Iran. Molecular characterization of isolated Staphylococcus spp. from diseased ewes were performed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and DNA sequencing of glyceraldehyde-3-phosphate dehydrogenase (gap) gene. Also, antibiotic resistance of staphylococcal isolates against different antibiotics was investigated. A total number of 900 milk samples from 450 native ewes in their mid-lactation period were examined by the California mastitis test (CMT). The CMT positive samples were cultured and bacteria were isolated from 86 (9.50%) glands and 74 (16.40%) ewes. The prevalence of subclinical mastitis in the examined ewes was 16.40%. Microbiological analysis of milk samples revealed that 27 out of 74 sheep with subclinical mastitis were infected with Staphylococcus spp. Amplification of gap gene of 27 Staphylococcus isolates generated a single amplicon of 933 bp in size confirming that isolates were belonged to Staphylococcus genus. Digestion of PCR products by AluI endonuclease generated different RFLP patterns for each species. Nucleotide sequencing of gap gene followed by phylogenetic analysis showed that the most dominant Staphylococcus species were S. epidermidis, S. xylosus and S. chromogenes. Staphylococcal isolates showed the highest resistance to penicillin and ampicillin. In conclusion, Staphylococcus species, except for the southern parts of the province, play an important role in the development of subclinical mastitis in sheep in West-Azerbaijan province of Iran. Also, chloramphenicol, ciprofloxacin and neomycin are the most effective antibiotics for treatment of this disease.
... Similar results were reported by Mohajeri et al. (2013) in Kermanshah, in western Iran, with a percentage of MRSA of 36% (17). Our results are in agreement with data collected by Salimnia and Brown (2005) among staphylococcus isolates in outpatient and inpatients at the Detroit medical center (DMC) and from Outreach specimens (18), and Malathi et al. in 2009, who found a percentage of 36.4% MRSA isolated from patients (19). The occurrence of MRSA results in this study was lower than some results in Iran and other countries. ...
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Background: Panton-Valentine leukocidin (luk-pv) is a cytotoxin that causes leukocyte destruction and tissue necrosis. Objectives: The aim of this study was to determine the prevalence of the pv1, femA, and mecA genes in staphylococcus aureus isolates from clinical specimens in hospitals in Ilam, Iran.
... Cycle sequencing reaction was carried out for the Rubella virus positive amplified products. The products were purified according to standard protocol, loaded onto ABI 3130 Genetic Analyzer (Applied Biosystem, USA) with polymer POP7 and sequences were analyzed as described earlier 17 . ...
... (iii) Nucleotide sequence analysis -Purification of PCR products was performed by adding 1 μl of 1U/ μl shrimp alkaline phosphatase (SAP) and 0.5 μl of 20 U/μl exonuclease (Exo) I (Fermentas, Life Science, USA) to 5μl of amplified product in a separate vial and then incubated at 37°C for 15 min followed by 85°C for 15 min. Exo-SAP-treated products were then subjected to cycle sequencing reaction as described earlier 27 Multiple sequence alignments were performed by using the CLUSTAL W algorithm 28 and were exported into the DAMBE programme 29 . Phylogenetic trees were constructed by the maximum-parsimony method 29 and the neighbour-joining method 29 . ...
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Background & objectives: There are only a few reports available on characterization of Propionibacterium acnes isolated from various ocular clinical specimens. We undertook this study to evaluate the role of P. acnes in ocular infections and biofilm production, and also do the phylogenetic analysis of the bacilli. Methods: One hundred isolates of P. acnes collected prospectively from ocular clinical specimens at a tertiary care eye hospital between January 2010 and December 2011, were studied for their association with various ocular disease conditions. The isolates were also subjected to genotyping and phylogenetic analysis, and were also tested for their ability to produce biofilms. Results: Among preoperative conjunctival swabs, P. acnes was a probably significant pathogen in one case; a possibly significant pathogen in two cases. In other clinical conditions, 13 per cent isolates were probably significant pathogens and 38 per cent as possibly significant pathogens. The analysis of 16S rRNA gene revealed four different phylogenies whereas analysis of recA gene showed two phylogenies confirming that recA gene was more reliable than 16S rRNA with less sequence variation. Results of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) had 100 per cent concordance with phylogenetic results. No association was seen between P. acnes subtypes and biofilm production. Interpretation & conclusions: RecA gene phylogenetic studies revealed two different phylogenies. RFLP technique was found to be cost-effective with high sensitivity and specificity in phylogenetic analysis. No association between P. acnes subtypes and pathogenetic ability was observed. Biofilm producing isolates showed increased antibiotic resistance compared with non-biofilm producing isolates.
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The clinical significance of many species belonging to coagulase-negative staphylococci (CoNS) other than Staphylococcus epidermidis has been increasingly recognized. The present study attempted the characterization of non-S. epidermidis CoNS isolates of ocular origin. The isolates were characterized phenotypically by analytical profile index (API) and genotypically by fluorescent amplified fragment length polymorphism (FAFLP). In addition, the presence of genes that are likely to enhance virulence such as biofilm related genes (icaA and icaB) and methicillin resistance (mecA) were detected. API identified seven different species with an identification score varying from 44% to 99%. S. haemolyticus and S. xylosus species identified by API showed good API scores when compared with all other species identified. FAFLP generated 12 clusters from all the isolates and appeared to be more discriminatory. API identification results corresponded well with the FAFLP results only in six clusters. Nearly 40% of isolates showed the presence of mecA and icaAB genes. Identification results of these two methods corresponded only in 48.4% of the isolates suggesting that the battery of tests using API were not sufficient enough to identify all the species of CoNS. Therefore, it is sensible to rely on two or more identification methods particularly when API species identification has a lower score. FAFLP genotyping appears to be a reliable and rapid alternative identification method for CoNS that can be used in conjunction with any other phenotypic method.
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Bacterial isolation, identification and antimicrobial Susceptibility tests were carried out in Ocular material collected with swab and polimethylmethacrylate (PMMA) or silicone intraocular lenses (IOL) from forty six patients submitted to cataract Surgery. Seventy six isolates and seven different microorganisms were identified. Coagulase-negative staphylococci (CNS) were the predominant microorganisms isolated from swabs (71.4% of cases), PMMA lenses (81.3%) and silicon lenses (77.8%). Coagulase-negative staphylococci isolates revealed high resistance to penicillin G followed by tetracycline, chloramphenicol and aminoglicosides. However, these isolates displayed great susceptibility to vancomycin, cephalothin and ofloxacin. Except for penicillin G Staphylococcus aureus was very sensitive to the antimicrobial agents including oxacillin. Among Gram-negatives, Proteus mirabilis was prevalent and presented high resistance to tetracycline and chloramphenicol. Enterococcus isolates were vancomycin sensitive.
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In order to identify methicillin-resistant staphylococci from clinical sources with ease and reliability, enzymatic detection of polymerase chain reaction (ED-PCR) was applied. ED-PCR is based on the capture of amplified products via biotin-streptavidin affinity and the detection of an incorporated hapten in amplified products with an enzyme-linked antibody. In order to identify methicillin-resistant staphylococci of all species, a 150-bp fragment of the mecA gene was targeted for ED-PCR. After PCR was performed with a pair of biotin and dinitrophenol 5'-labeled primers, the reaction mixture was applied to a microtiter well precoated with streptavidin. Thereafter, bound PCR products were detected colorimetrically with alkaline phosphatase-conjugated anti-dinitrophenol antibody. The extraction of DNA from staphylococcal cells for PCR was simplified so that it could be performed within one tube. The total assay, including PCR, took less than 3 h. The sensitivity of mecA gene detection ranged from greater than 5 x 10(2) CFU per tube for Staphylococcus aureus to greater than 5 x 10(3) CFU per tube for Staphylococcus epidermidis. Genotyping results obtained by ED-PCR of 161 tested strains from the colonies (97 strains of S. aureus and 64 strains of coagulase-negative staphylococci) were compared with the phenotypic susceptibilities of the strains to oxacillin. The results of ED-PCR showed excellent agreement with the MICs of oxacillin with very few exceptions; only one strain of S. aureus and two strains of coagulase-negative staphylococci were found to possess the mecA gene, which was discrepant with their phenotypes. Fifty-five blood culture samples were also tested by ED-PCR. For staphylococcal isolates in 33 of the cultures, oxacillin MICs were >4 microgram/ml; 31 of the 33 staphylococcal isolates were determined by ED-PCR to be mecA gene positive. These results suggest that ED-PCR can be used with reasonable confidence in the clinical microbiological laboratory.
Infectious endophthalmitis is a devastating complication of cataract surgery resulting in significant loss of visual function in many cases. Early diagnosis together with appropriate therapy is therefore essential in order to optimise visual outcome. This article reviews the incidence, aetiology, risk factors, clinical presentation, microbiology, management, outcome and prevention of bacterial endophthalmtis following cataract surgery. Special emphasis is placed on how to perform a vitreous biopsy and inject intravitreal antibiotics.
AIMS: To identify and determine antibiotic susceptibility of coagulase negative staphylococci (CoNS) isolated from patients with chronic blepharitis, purulent conjunctivitis, and suppurative keratitis. METHODS: A retrospective review of all culture positive cases of chronic blepharitis, purulent conjunctivitis, and suppurative keratitis between July 1995 and December 1996 was performed. Cases in which CoNS were the sole isolates were analysed. Species identification was performed by using a commercially available standardised biochemical test system. Antibiotic susceptibility to penicillin, gentamicin, tetracycline, erythromycin, ciprofloxacin, and teicoplanin was determined by agar disc diffusion (Kirby-Bauer method). Teicoplanin resistance was confirmed by agar dilution. RESULTS: 42 Staphylococcus epidermidis, four S warneri, three S capitis, two S hominis, one each of S xylosus, S simulans, S equorum, and S lugdunensis were identified. 37 CoNS were penicillin resistant, 12 gentamicin resistant, 28 tetracycline resistant, 18 erythromycin resistant, four ciprofloxacin resistant, and one teicoplanin resistant (MIC, 32 microg/ml). In total, 16 strains were resistant to three or more antibiotics. CONCLUSION: Species of CoNS apart from S epidermidis may be isolated from patients with corneal and external infection. Antibiotic susceptibility of CoNS is unpredictable and multiresistant strains are common. As a result, antibiotic susceptibility testing should be performed in all cases of clinically significant ocular infections caused by CoNS.
Cataracts are the most common cause of blindness in the world and cataract surgery is one of the most common surgical procedures performed. During surgery the opacified crystalline lens is removed and replaced with a lens implant. Bacterial infection of the internal eye (bacterial endophthalmitis) following cataract surgery appears to have increased since the 1990s. The infection is the result of inoculation of the patient’s bacterial flora into the eye during surgery. Under normal circumstances a small inoculum of bacteria can be cleared from the eye without clinical infection. In this chapter, we discuss many factors that can predispose a patient to developing a clinical endophthalmitis, including those related to the lens implant and host response. There is evidence to suggest that the intraocular lens may provide a niche where bacteria are protected from the host mechanisms that normally clear bacteria from the eye.
Purpose: To analyse commonly used ocular antibiotics and determine their in-vitro efficacies against bacterial keratitis pathogens. Methods: A retrospective review of microbiology records at the LV Prasad Eye Institute in Hyderabad, India identified 1,633 bacterial keratitis isolates. Antibiotic susceptibility of corneal isolates was determined for various ocular antibiotics using the Kirby-Bauer disc-diffusion method. Results: Cefazolin had coverage against 1,296 (83.0%) of 1,562 isolates tested; chloramphenicol against 1,136 (71.7%) of 1,585 isolates; ciprofloxacin against 1,080 (69.3%) of 1,558 isolates; gentamicin against 1,106 (70.6%) of 1,567 isolates; norfloxacin against 1,057 (67.7%) of 1,561 isolates; vancomycin against 463 (84.3%) of 549 isolates; and framycetin against 105 (36.2%) of 290 isolates. Also included is a breakdown by species, and sensitivity profiles for resistant isolates.Conclusion: This study provides information on the efficacies of ocular antibiotics commonly used against bacterial keratitis pathogens. It also examines the antibiotic susceptibility profiles for corneal pathogens that are resistant to an ocular antibiotic but sensitive to other selected antibiotics. It is hoped that this information will aid in the decision-making of empiric initial treatment of bacterial keratitis.
A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.
Methicillin resistance in staphylococci is determined by mec, composed of 50 kb or more of DNA found only in methicillin-resistant strains. mec contains mecA, the gene for penicillin-binding protein 2a (PBP 2a); mecI and mecR1, regulatory genes controlling mecA expression; and numerous other elements and resistance determinants. A distinctive feature of methicillin resistance is its heterogeneous expression. Borderline resistance, a low-level type of resistance to methicillin exhibited by strains lacking mecA, is associated with modifications in native PBPs, beta-lactamase hyperproduction, or possibly a methicillinase. The resistance phenotype is influenced by numerous factors, including mec and beta-lactamase (bla) regulatory elements, fem factors, and yet to be identified chromosomal loci. The heterogeneous nature of methicillin resistance confounds susceptibility testing. Methodologies based on the detection of mecA are the most accurate. Vancomycin is the drug of choice for treatment of infection caused by methicillin-resistant strains. PBP 2a confers cross-resistance to most currently available beta-lactam antibiotics. Investigational agents that bind PBP 2a at low concentrations appear promising but have not been tested in humans. Alternatives to vancomycin are few due to the multiple drug resistances typical of methicillin-resistant staphylococci.
The presence and sequences of genes that regulate the expression of methicillin resistance was investigated in 42 isolates of Staphylococcus aureus and 102 isolates of coagulase-negative staphylococci (CNS). PCR was used to detect mecA and the regulatory genes mecR1 and mecI. In a selected group of isolates, the sequences of mecI and the mec promoter region were also determined and compared with the sequences obtained from pre-MRSA strain N315. The genetic diversity of the collection was assessed by pulsed-field gel electrophoresis (PFGE). mecA was present in 21 S. aureus and 44 CNS. mecR1 was associated with mecA in all S. aureus and in all CNS, except two isolates of Staphylococcus haemolyticus. mecI was present in 48% of mecA-positive S. aureus and 50% of mecA-positive CNS. In six S. aureus isolates, mecI contained a termination codon at nucleotide 202 which would truncate the MecI protein. No mutation was found in the mecI gene of the four other S. aureus and 15 CNS sequenced. Seven isolates of Staphylococcus simulans had a single nucleotide substitution in the mec promoter region. Expression of methicillin resistance could be explained for all mecA-positive staphylococci with mutations within mecI or in the mec promoter region or in which mecI was deleted. However, the 'wild type' sequences observed in four S. aureus and eight CNS suggest that there is another mechanism for overcoming the repression of resistance caused by mecI.
Ninety-nine clinical staphylococcal isolates (58 coagulase-negative Staphylococcus spp. [CoNS] and 41 Staphylococcus aureus isolates) were evaluated for susceptibility to oxacillin. The following susceptibility testing methods, media, and incubation conditions were studied: agar dilution by using Mueller-Hinton (MH) medium (Difco) supplemented with either 0, 2, or 4% NaCl and incubation at 30 or 35 degrees C in ambient air for 24 or 48 h; disk diffusion by using commercially prepared MH medium (Difco) and MH II agar (BBL) and incubation at 35 degrees C in ambient air for 24 or 48 h; and agar screen (spot or swab inoculation) by using commercially prepared agar (Remel) or MH agar (Difco) prepared in-house, each containing 4% NaCl and 6 microg of oxacillin/ml (0.6-microg/ml oxacillin was also studied with MH agar prepared in-house for the agar swab method and CoNS isolates) and incubation at 35 degrees C in ambient air for 24 or 48 h for swab inoculation and at 30 or 35 degrees C in ambient air for 24 or 48 h for spot inoculation. The results for these methods were compared to the results for mecA gene detection by a PCR method. Given the ability to support growth and the results for susceptibility testing (the breakpoint for susceptible isolates was </=2 microg/ml), the best methods for CoNS isolates were (i) agar dilution by using MH medium supplemented with 4% NaCl and incubation at 35 degrees C for 48 h (no growth failures were noted, and sensitivity was 97.6%) and (ii) agar screen (swab inoculation) by using MH medium prepared in-house supplemented with 4% NaCl and containing 0.6 microg oxacillin/ml and incubation at 35 degrees C for 48 h (one isolate that did not carry the mecA gene did not grow, and the sensitivity was 100%). All but one (agar dilution without added NaCl and incubation at 30 degrees C for 48 h) of the methods tested revealed all oxacillin-resistant S. aureus isolates, and no growth failures occurred with any method. If the breakpoint for susceptibility was lowered to </=1 microg/ml for agar dilution methods, more CoNS isolates with oxacillin resistance related to the mecA gene were detected when 0 or 2% NaCl agar supplementation was used. Only one CoNS isolate with mecA gene-associated resistance was not detected by using agar dilution and MH medium supplemented with 4% NaCl with incubation for 48 h. When the breakpoint for susceptibility was decreased 10-fold (from 6.0 to 0.6 microg of oxacillin per ml) for the agar swab screen method, fully 100% of the CoNS isolates that carried the mecA gene were identified.