Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

Users Online : 41258

AbstractMaterial and MethodsResultsDiscussionKey MessageReferencesDOI and Others
Readers' Comments (0) Article in PDF Audio Visual Citation Manager Article Statistics Link to PUBMED Print this Article Send to a Friend
Advertisers Access Statistics Resources

Dr Archana Dambal

"Journal of clinical and diagnostic research is a welcome change in publishing practices. It aims to reach out to the grass-root level researchers who do not lack in experience, clinical material and ideas, but lack in their knowledge in English language and statistics. The journal achieves it's aim by supporting in these exact domains.
It also gives due credit to all research designs like descriptive and qualitative studies while many journals ignore these important study designs. The rigorous review process does not allow any compromise in quality
It is indexed in many indexing agencies and the articles are available under creative commons licence free of cost
The frequency of publication supports many aspiring authors from India and other countries.
It's wide scope welcomes articles across various specialities in medicine. In an era when there is an unscientific insistence on speciality specific research by regulatory bodies in medical education, JCDR supports collaborative research across specialities. I wish the publisher all the best in his future endeavors."



Dr. Archana Dambal
Department of General Medicine,
Belgaum Institute of Medical Sciences,Belgaum, Karnataka,INDIA,
On 30 Nov 2018




Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011

Important Notice

Original article / research
Year : 2011 | Month : November | Volume : 5 | Issue : 7 | Page : 1359 - 1362

Comparison of Various Phenotypic Methods and mecA Based PCR for the Detection of MRSA

Pramodhini S., Thenmozhivalli P.R., Selvi R., Dillirani V., Vasumathi A., Agatha D.

Department of Microbiology, Stanley Medical College and Hospital, Chennai, India

Correspondence Address :
Pramodhini S.
Assistant Professor
Department of Microbiology,
Mahatma Gandhi Medical College and Research Institute,
Pillaiyarkuppam, Pondicherry – 607 402
Phone: +91 9445216145
E-mail: pramo4@yahoo.co.in

Abstract

Background: Methicillin resistant Staphylococcus aureus (MRSA) is the most commonly emerging pathogen in community and hospital acquired infections. Hence, an accurate detection is not only important for the control of the infection, but also to control the endemicity of MRSA.

Aim: To evaluate the efficacy of various phenotypic methods with mecA based PCR to detect MRSA. We also studied the resistance pattern of the MRSA isolates. Settings and Design: This was a prospective study which was conducted at a tertiary care hospital.

Materials and Methods: A total of 55 S. aureus strains which were isolated from patients with superficial and deep abscesses were included in this study. Methicillin resistance which was determined by oxacillin disc diffusion, cefoxitin disc diffusion and the oxacillin screen agar test was compared with mecA based PCR.

Results: Among the 55 S . aureus isolates, 20 (36.4%) isolates were positive for the mecA gene by PCR. Both the cefoxitin disc diffusion and the oxacillin screen agar test showed 100% sensitivity and 100% specificity, while oxacillin disc diffusion showed 90% sensitivity and 100% specificity. The resistance percentage of the MRSA isolates to erythromycin, ciprofloxacin and amikacin were 80%, 30% and 25%, respectively.

Conclusion: Conventional MRSA detection assays like the cefoxitin disc diffusion test and the oxacillin screen agar test are simple and relatively cheap and can be used as alternatives to PCR for the detection of MRSA in resource constraint settings. Also, most of the MRSA strains in this study showed coresistance to many classes of antibiotics and thus they qualified as multi-drug resistant S. aureus.

Keywords

MRSA, cefoxitin disc diffusion, oxacillin screen agar, mecA gene

How to cite this article :

Pramodhini S., Thenmozhivalli P.R., Selvi R., Dillirani V., Vasumathi A., Agatha D.. COMPARISON OF VARIOUS PHENOTYPIC METHODS AND MECA BASED PCR FOR THE DETECTION OF MRSA. Journal of Clinical and Diagnostic Research [serial online] 2011 November [cited: 2019 Aug 21 ]; 5:1359-1362. Available from
http://www.jcdr.net/back_issues.asp?issn=0973-709x&year=2011&month=November&volume=5&issue=7&page=1359-1362&id=1659

INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) strains were first identified in 1961, immediately after the introduction of methicillin in the clinical settings. Subsequently, an increase in the resistance to methicillin among the S. aureus isolates has been observed globally (1). MRSA is one of the major pathogens which are associated with serious nosocomial infections, because these strains generally show multiple drug resistance which limits the treatment possibilities. MRSA has become established outside the hospital environment and it is now appearing in community populations without any identifiable risk factors (2).

The methicillin resistance in Staphylococci is due to the acquisition of the mecA gene, which encodes the low-affinity penicillin-binding protein 2a (3). The presence of the mecA gene in S. aureus defines methicillin resistance, while the absence of the gene indicates methicillin susceptibility (4). Methicillin resistance can be difficult to detect, because the mecA-positive strains can differ in their level of expression of resistance. The resistance is typically heterogeneous, with only a few cells (one in 104 or 106) expressing the phenotype.

The mecA gene is highly conserved among the Staphylococcal species and therefore, presently, the detection of this gene by the polymerase chain reaction (PCR) is considered as the “goldstandard” for the detection of methicillin resistance in Staphylococci (5),(6),(7). While PCR is considered as the gold standard assay for the detection of methicillin resistance, it still remains a time-consuming and expensive method; besides, it is not available in most of the routine laboratories.

The objective of the present study was to determine the methicillin resistance in S. aureus by mecA based PCR and to evaluate the usefulness of various phenotypic methods for the detection of MRSA in comparison to mecA based PCR. We also aimed to study the resistance pattern of the MRSA isolates.

Material and Methods

Clinical Isolates
A prospective study was conducted over a period of one year from May 2008 to June 2009 at a tertiary care hospital in south India. A total of 55 S. aureus strains which were isolated from patients with superficial and deep abscesses were included in this study. All the isolates were identified as S. aureus by their colony morphology, gram staining and their catalase and coagulase tests (both tube and slide tests). These 55 clinical isolates were tested for methicillin resistance by oxacillin disk diffusion, cefoxitin disc diffusion and the oxacillin screen agar test. From this collection, all the isolates were evaluated further by using mecA based PCR.

All the Staphylococcal isolates were tested for susceptibility to a predetermined battery of antibiotics by the Kirby-Bauer disc diffusion method. The antibiotics which were tested included Penicillin (10u), Ampicillin (10μg), Cefotaxime (30μg), Amikacin (30 μg), Erythromycin (15 μg), Ciprofloxacin (5μg) Oxacillin (1μg) and Vancomycin (30 μg). The zones of inhibition were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines (8).

Phenotypic Detection of MRSA
The Oxacillin D isk Diffusion Method: The Oxacillin disk (1 μg) diffusion method was carried out on Mueller-Hinton agar which was supplemented with 2% NaCl to detect MRSA according to the CLSI guidelines (8). The plates were incubated at 35°C and the results were recorded after 24 hrs of incubation. The isolates were considered as resistant when the diameter of inhibition was ≤10 mm, as intermediately resistant when the diameter was 11-12 mm and as sensitive when the diameter was ≥13 mm (8).

The Cefoxitin Disc Diffusion Test:
The Cefoxitin disc diffusion method was carried out on Mueller-Hinton agar by using a 30 μg cefoxitin disc. An inhibition zone diameter of ≤ 21 mm was reported as methicillin resistant and a diameter of ≥ 22 mm was considered as methicillin sensitive (8).

The Oxacillin Screen Agar Test:
Muller-Hinton agar plates containing 4% NaCl and 6 μg/ml of oxacillin were prepared. To perform the oxacillin screen test, a swab which was dipped in 0.5 Mc Farland’s suspension of the isolate was deposited as a spot on the agar surface and it was incubated at 35°C for 24 h. The plates were observed carefully in transmitted light for any growth. Any growth after 24 hr was interpreted as oxacillin resistance (9).

The Genotypic Detection of MRSA
Detection of the mecA Gene by Polymerase Chain Reaction: Bacterial DNA was obtained by the rapid cell lysis method as described by Unal et al (10). For the DNA extraction, 0.1 mL of an overnight culture of bacteria in Mueller-Hinton broth washarvested by centrifuging the broth in a microcentrifuge tube at 16,000×g for 30 seconds. The precipitates were resuspended in 50 μL lysostaphin (100 μg/mL; Sigma) and they were incubated at 37°C for 10 minutes. Following the addition of 50 μL proteinase K (100 μg/mL; Sigma Aldrich) and 150 μL 100 mm Tris (pH 7.5), the suspension was incubated for 10 minutes at 37°C and then boiled for 5 minutes. After centrifugation at 13,000×g for 2 minutes, the supernatant which contained the extracted bacterial DNA was used in the PCR assay.

From this suspension, a 5μL volume was directly used as the template for the PCR amplification of the mec A gene fragments. The mec A1 (5´ – GTA GAA ATG ACT GAA CGT CCG ATA A – 3´) and the mec A2 (5´ – CCA ATT CCA CAT TGT TTC GGT CTA A – 3´) primers were used for the amplification of the 310 bp fragment of the methicillin-resistant gene ( mec A) (11). A 50 μl PCR reaction consisted of plus 45 μl of the master mix which contained the PCR buffer (1×), dNTP mix (0.2 mM of each), the primer (0.5 μM), Taq DNA polymerase (0.25 U), and MgCl 2 (1.5 mM) with 5 μL of the template DNA. The cycling conditions were as follows: 4 minutes at 94°C, followed by 30 cycles of denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds, extension at 72°C for 1 minute and the final extension step at 72°C for 3 minutes. The PCR products were visualized on an 0.8% agarose gel with ethidium bromide dye under a UV transilluminator. Amplicons of 310 bp were consistent with the mecA gene amplification.

Quality control
The quality control strains – methicillin sensitive S. aureus (MSSA) ATCC 25923 and methicillin resistant S. aureus (MRSA) ATCC 43300 – were used as the negative and positive controls, respectively.

Statistical Analysis
The percentages were calculated for the categorical variables. The sensitivity, specificity and the positive and negative predictive values were calculated by using the GraphPad InStat version 3.00 for Windows 95 and the GraphPad Software (San Diego, CA, USA) for determining the diagnostic value of the various phenotypic methods for detecting methicillin resistance.

Results

Among the 55 S . aureus isolates, 20 (36.4%) were positive for the mecA gene by PCR. Methicillin resistance was detected by oxacillin disc diffusion, cefoxitin disc diffusion and the oxacillin screen agar test in 18, 20 and 20 isolates, respectively. The sensitivity, specificity and the positive and negative predictive values of the various phenotypic methods in comparison to PCR, for the detection of MRSA, are summarized in (Table/Fig 1).

In our study, the resistance percentage of the MRSA isolates were as follows: penicillin- 100%, ampicillin- 85%, cephotaxime and erythromycin- 80%, ciprofloxacin- 30%, amikacin- 25% and all the strains were 100% sensitive to vancomycin.

Discussion

S. aureus is one of the most common causes of nosocomial as well as community-acquired infections. Methicillin resistance (as a result of the mecA gene which encodes the additional penicillin binding protein, PBP2a) renders S. aureus resistant to all the β-lactam antibiotics which is the most important group of antibiotics in the treatment of Staphylococcal infections.

The recent increase in the methicillin-resistant and ultipleresistant strains at large hospitals have started to pose a great difficulty in selecting antimicrobial agents for the management of the infections that they cause. Hence, an accurate and rapid detection of methicillin resistance in Staphylococci is therefore important, not only for choosing the appropriate antibiotic therapy for the individual patient, but also for the control of the endemicity of the MRSA.

Our study revealed that, overall the rate of methicillin-resistance with S. aureus was nearly 36.4%. Similar rates which were reported in other studies from tertiary care centers all over the world (12),(13) supported this high incidence which was found in our study.

In our study, the oxacillin disc diffusion method detected 18 of the 20 cases of MRSA with a sensitivity of 90% and a specificity of 100%, which was in concordance to the results of the previous study (14), which showed 87.5% sensitivity and 100% specificity by the oxacillin disc diffusion method. Although the oxacillin disc diffusion test has high sensitivity and specificity for detecting methicillin resistance, some difficulties have been encountered with it in detecting the hetero-resistant isolates of S. aureus. The accurate determination of methicillin resistance in Staphylococci by the oxacillin disc diffusion method may be affected by various components of medium, temperature, and the duration of incubation (15). Hence, other phenotypic methods like the agar screen method and the cefoxitin disc diffusion method have been evaluated.

MRSA detection by the oxacillin screen agar method identified all the 20 MRSA with 100% sensitivity and 100% specificity. A similar study reported that the sensitivity of this method approached 100% for the detection of MRSA and 95% for the coagulase-negative S. aureus (16).

In this study, the cefoxitin disc diffusion method detected 20 (36.4%) out of the 55 isolates as MRSA, which accounted for 100% sensitivity and specificity as compared to the mecA-based PCR. Cefoxitin, a cephamycin, is a more potent inducer of the mecA regulatory system and an accurate surrogate marker for the detection of MRSA in the routine susceptibility testing. It has been suggested that no special medium or incubation temperature is required for cefoxitin as is required for oxacillin (17). Recent studies have indicated that disc diffusion testing by using the cefoxitin disc is far superior to most of the currently recommended phenotypic methods like oxacillin disc diffusion and oxacillin screen agar testing and that it is now an accepted method for the detection of MRSA by many reference groups including CLSI (18).

In our study which was conducted to determine the resistance pattern of the MRSA isolates, the resistance to erythromycin, amikacin and ciprofloxacin were 85%, 40% and 30% respectively and no strains were found to be resistant to vancomycin, which was similar to the findings of several other studies (19),(20). The MRSA showed a high level of resistance to most of the antibiotics in comparison to the methicillin sensitive S. aureus. Also, most of the MRSA in this study showed co-resistance to many classes ofantibiotics at the same time and thus they qualified as multi-drug resistant S. aureus (MDR-MRSA).

In conclusion, as has been shown in this as well as other studies, the oxacillin disk diffusion method was found to be less sensitive for the detection of MRSA. The results of the cefoxitin disc diffusion test and the oxacillin screen agar test were in concordance with the results of PCR for the mecA gene. Moreover, these conventional MRSA detection assays like the cefoxitin disc diffusion test and the oxacillin screen agar test are simple and relatively cheap and they can be used as an alternative to PCR for the detection of MRSA in resource constraint settings.

Key Message

‘MRSA’ is a term which is used to describe the Staphylococcus aureus isolates that are resistant to all the available β-lactam antibiotics, including the penicillins and the cephalosporins. n Methicillin resistance in S. aureus is primarily mediated by the mecA gene, which codes for the modified penicillin-binding protein 2a (PBP 2a or PBP 2’) Strains that possess the mecA gene are either heterogeneous or homogeneous in their expression of resistance. The phenotypic expression of the resistance can vary, depending on the growth conditions (e.g. temperature, osmolarity and culture medium supplements such as NaCl or sucrose) The detection of the mecA gene by PCR is considered as the gold standard.

References

1.
Brown DFJ. Detection of methicillin/oxacillin resistance in Staphylococci. J Antimicrob Chemother 2001; 48:65-70
2.
Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, Boyle-Vavra S, Community-acquired methicillin- resistant Staphylococcus aureus in children with no identified predisposing risk. JAMA 1998; 279 :593-98.
3.
Hartman BJ, Tomasz A. Low-affinity penicillin-binding protein which is associated with β-lactam resistance in Staphylococcus aureus. J Bacteriol 1984;158:513-16.
4.
Gradelski E, Valera L, Aleksunes L, Bonner D, Fung-Tomc J. Correlation between the genotype and phenotypic categorization of staphylococci based on the methicillin susceptibility and resistance. J Clin Microbiol 2001;39:2961-63.
5.
Bignardi GE, Woodford N, Chapman A, Johnson AP, Speller DC. Detection of the mecA gene and the phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or lowlevel methicillin resistance. J Antimicrob Chemother 1996;37:53-63
6.
Maes N, Magdalena J, Rottiers S, De Gheldre Y, Struelens MJ. Evaluation of a triplex PCR assay to discriminate Staphylococcus aureus from coagulase-negative staphylococci and to determine methicillin resistance from blood cultures. J Clin Microbiol 2002;40:1514-17
7.
Murakami K, Minamide W, Wada K, Nakamura E, Teraoka H, Watanabe S. Identification of the methicillin-resistant strains of staphylococci by the polymerase chain reaction. J Clin Microbiol 1991;29:2240-44.
8.
Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twentieth informational supplement. CLSI document M100-S20. Wayne, PA: CLSI; 2006.
9.
Skov R, Smyth R, Clausen M, Larsen AR, Frimodt-Møller N, Olsson- Liljequist B, et al. Evaluation of a cefoxitin 30 μg disc on Iso-Sensitest agar for the detection of methicillin- resistant Staphylococcus aureus. J Antimicrob Chemother 2003;52:204-07.
10.
Unal S, Hoskins J, Flokowitsch JE, Wu CY, Preston DA, Skatrud PL. Detection of Methicillin-resistant staphylococci by using the polymerase chain reaction. J Clin Microbiol 1992;30:1685-91.
11.
Perez-Roth E, Claverie-Martin F, Villar J, Mendez-Alvarez S. Multiplex PCR for the simultaneous identification of Staphylococcus aureus and the detection of methicillin and mupirocin resistance. J Clin Microbiol 2001;39:4037-41.
12.
Thompson RL, Cabezuclo I, Werizel RP. Epidemiology of the nosocomial infections which are caused by methicillin -resistant Staphylococcus aureus. Ann Intern Med 1982; 97: 309- 17
13.
Cafterkey MT, Hone R, Keane CT. Antimicrobial chemotherapy of septicaemia due to methicillin – resistant Staphylococcus aureus. Antimicrob Agents Chemother 1985; 28: 819-23.
14.
Anand KB, Agrawal P, Kumar S, Kapila K. Comparison of the cefoxitin disc diffusion test, the oxacillin screen agar test and PCR for the mecA gene for detection of MRSA. Indian J Med Microbiol 2009;27: 27-29.
15.
Fernandes CJ, Fernandes LA, Collignon P. Cefoxitin resistance as a surrogate marker for the detection of methicillin-resistant Staphylococcus aureus . J Antimicrob Chemother 2005;55:506-10.
16.
Chambers HF. Methicillin resistance in Staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev 1997;10:781–91
17.
Brown DFJ, Edwards DI, Hawkey PM, Morrison D, Ridgway GL et al. Guidelines for the laboratory diagnosis and the susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA ). J Antimicrob Chemother 2005; 56:1000-18.
18.
Sakoulas G, Gold HS, Venkataraman L, DeGirolami PC, Eliopoulos GM, Qian Q. Methicillin-resistant Staphylococcus aureus: Comparisonof the susceptibility testing methods and the analysis of the mecApositive susceptible strains. J Clin Microbiol 2001;39:3946-51.
19.
Mohanasoundaram KM, Lalitha MK. Comparison of the phenotypic versus genotypic methods in the detection of methicillin resistance in Staphylococcus aureus Indian J Med Res 2008;127:78-84.
20.
Vidhani S, Mehndiratta PL, Mathur MD. Study of methicillin resistant S. aureus (MRSA) isolates from high risk patients. Indian J Med Microbiol 2001;19: 87-90.

Tables and Figures
[Table / Fig - 1]
DOI and Others

JCDR/2011/1659

JCDR is now Monthly and more widely Indexed .
  • Emerging Sources Citation Index (Web of Science, thomsonreuters)
  • Index Copernicus ICV 2017: 134.54
  • Academic Search Complete Database
  • Directory of Open Access Journals (DOAJ)
  • EBSCOhost
  • Google Scholar
  • HINARI Access to Research in Health Programme
  • Indian Science Abstracts (ISA)
  • Journal seek Database
  • Google
  • Popline (reproductive health literature)
  • www.omnimedicalsearch.com