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

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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
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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.
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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 : 2009 | Month : December | Volume : 3 | Issue : 6 | Page : 1915 - 1920

Detection Of Biofilm Producing Staphylococci: Need Of The Hour

BOSE S *, KHODKE M **, BASAK S ***, MALLICK S K ****,

*,(M.D)Professor of Microbiology,**search Assistant, Deptt of Microbiology,***(M.D)Professor of Microbiology,****(M.B.B.S)Tutor,Deptt. of Microbiology,Jawaharlal Nehru Medical College,Wardha(M.S)

Correspondence Address :
Dr. S. Bose,Professor of Microbiology J. N. Medical College,Sawangi (M), Wardha-442004 (M.S.)E-mail: drseema11ghosh@gmail.com,drseema11ghosh@hotmail.com

Abstract

Biofilms are group of microorganisms encased in an exopolymeric coat. They have been associated with a variety of persistent infections that respond poorly to conventional antibiotics. In this study detection of biofilm productions by Staphylococcus spp. was done by using Congo red agar (CRA) methods, tube methods (TM) and tissue culture plate (TCP) methods. Out of 179 Staphylococcus spp., 111 were S.epidermidis and 68 were S.aureus. 44.69% of S.epidermidis and 32.96% S.aureus were slime producers. 97 isolates were detected as slime producer by TCP method, 76 by TN and 11 by CRA method. High resistances to conventional antibiotics were shown by biofilm producers.
This study summarized the prevalence, antibiotic sensitivity pattern and suitable and reproducible method for detection of biofilm producing Staphylococci.

Keywords

Biofilm detection, Staphylococci, Congo red agar, Antibiotic resistance

Introduction
Biofilms are a group of microorganisms attached to a surface and covered by an exopolysaccharide matrix. Various changes occur during their transition from planktonic to a surface attached community. In response to certain environmental signals, new phenotypic characteristics develop in such bacteria. The first recorded observation concerning biofilm was probably given by Henrici in 1933, who observed that water bacteria are not free floating but grow upon submerged surfaces (1). Certain surface protein, extracellular proteins, capsular polysaccharides, adhesins (PS/A) and autolysin (encoded by atIE gene) are involved in regulation of biofilm production. The ica gene codes for intracellular adhesion (ICA) and may also code for TS/A and is required for biofilm production (1),(2),(3).

Biofilms are often site for quorum sensing influencing their formation. Availability of key nutrients, chemotaxis towards surface, motility of bacteria, surface adhesins and presence of surfactants are certain factors which influence biofilm formation.(3),(4). Using Bacillus subtilis from soil, Dr Stanley Wall has shown that a protein called Deg U helps the individual bacteria to decide whether to form a biofilm or not (5). Biofilm producing Staphylococci frequently colonize catheters and medical devices and may cause foreign body related infections. They easily get attached to polymer surfaces.(4),(5),(6) Crampton et al showed that like S epidermidis, S aureus also has ica locus encoding the function of intracellular adhesion and biofilm formation (7). According to a recent public announcement from National Institute Of Health, more than 60% of all infections are caused by biofilm (8). Biofilm organisms have an inherent resistance to antibiotics, disinfectants and germicides. The use of synthetic material for implantation is widely associated with “Implant associated infection” due to biofilm production. In the long run they may be very damaging because of immune complex disease (2),(9),(10).

Aims & Objectives
Keeping all these things in mind, the present study was undertaken to detect the prevalence of biofilm producer and nonproducer Staphylococci isolated from clinical materials in our laboratory by three different methods, viz. tissue culture plate (TCP) method, tube method (TM) and Congo red agar (CRA) method and to compare the above mentioned three different methods for biofilm production.

Material and Methods

A total of 179 clinical isolates of Staphylococci spp. were isolated from blood, infected devices, skin surface, urine, pus etc. from Indoor patient department (IPD) of a rural hospital with tertiary care in Central India over a period of 1 year. Isolates were identified by Gram staining, catalase and coagulase tests. Reference strains of Staphylococcus epidermidis ATCC 35984 (high slime producer),

ATCC35983 (moderate slime producer) and ATCC 12228 (nonslime producer) were also included in this study (11). Detection of biofilm production of 179 Staphylococci spp. was done by following three methods.

1. Tissue culture plate (TCP) method (8),(11)
2. Tube method (TM) (11),(12)
3. Congo red agar (CRA) method (11),(13)

1. Tissue Culture Plate Method
10 ml of Trypticase soy broth with 1% glucose was inoculated with a loopful of test organism from overnight culture on nutrient agar. The broth was incubated at 370C for 24 hours. The culture was further diluted 1:100 with fresh medium. 96 wells flat bottom tissue culture plates were filled with 0.2 ml of diluted cultures individually. Only sterile broth was served as blank. Similarly control organisms were also diluted and incubated. All three controls and blanks were put in the tissue culture plates. The culture plates were incubated at 370C for 24 hours. After incubation, gentle tapping of the plates was done. The wells were washed with 0.2 ml of phosphate buffer saline (pH 7.2) four times to remove free floating bacteria. Biofilms which remained adherent to the walls and the bottoms of the wells were fixed with 2% sodium acetate and stained with 0.1% crystal violet. Excess stain was washed with deionized water and plates were dried properly. Optical densities (OD) of stained adherent biofilm were obtained with a micro ELISA autoreader at wave length 570 nm. Experiment was performed in triplicate and repeated thrice. Average of OD values of sterile medium were calculated and subtracted from all test values (8),(11).

2. Tube Method
10 ml Trypticase soy broth with 1% glucose was inoculated with a loopful of test organism from overnight culture on nutrient agar individually. Broths were incubated at 370C for 24 hours. The cultures were decanted and tubes were washed with phosphate buffer saline (pH 7.3). The tubes were dried and stained with 0.1% crystal violet. Excess stain was washed with deionized water. Tubes were dried in inverted position.

In positive biofilm formation, a visible stained film was seen lining the wall and bottom of the tube. Experiments were done in triplicate for 3 times and read as absent, weak, moderate and strong.(11),(12)

3. Congo Red Method
The medium composed of Brain heart infusion broth (37 gm/l), sucrose (5 gm/l), agar number 1 (10 gm/l) and Congo red dye (0.8 gm/l). Congo red stain was prepared as concentrated aqueous solution and autoclaved at 1210C for 15 minutes. Then it was added to autoclaved Brain heart infusion agar with sucrose at 550C. Plates were inoculated with test organism and incubated at 370C for 24 to 48 hours aerobically. Black colonies with a dry crystalline consistency indicated biofilm production (11),(13).

Antibiotic sensitivity test was done on Muller-Hinton agar (MHA) using following antibiotic discs- penicillin (10units), ampicillin(10µg), ofloxacin(5µg), ciprofloxacin (5µg), cefotaxime(30µg), erythromycin(15µg), co-trimoxazole(25µg), amikacin(30µg), gentamicin(10µg), Netillmicin(30µg), linezolid(30µg), vancomycin(30µg), Antibiotics discs were procured from HiMedia Laboratories Pvt. Ltd, India. ATCC Staphylococcus aureus 25922 was used as control. Antibiotic sensitivity test was done as per Kirby-bauer disc diffusion method. (14)

Results

(Table/Fig 1) A total of 179 Staphylococci were isolated from various clinical materials. Out of 179 Staphylococcus spp. 111 S.epidermidis and 68 S.aureus. Among 111 S.epidermidis isolated from different clinical samples, 44.69% were slime producers and 17.32% non-slime producers, whereas among 68 S. aureus, 32.96% were slime producers and 5.03% were non-slime producers. Among S.epidermidis maximum biofilm producers were from catheter (51 out of 111). Among 51 catheters from which S.epidermidis were isolated, 40 were intravenous catheter and 11 were foley’s catheters. From two of such catheters adherent slimy growth were seen. Maximum numbers of biofilm producing S.aureus were from orthopedic implants (19 out of 68). We found high resistance pattern among biofilm producers in comparison with non-biofilm producers. Two strains of S. aureus were intermediate Vancomycin sensitive. Both the strains were biofilm producers (Table/Fig 2).

(Table/Fig 3) OD value of stained adherent biofilm was obtained with a microELISA autoreader at wave length 570nm. OD value less than 0.120 was considered as non-biofilm producers, 0.120 -0.240 as moderate biofilm producers and more than 0.240 as strong biofilm producers.

(Table/Fig 4) Among 179 clinical isolates of Staphylococci, 15.64% were high biofilm producers by TCP methods,12.30% by TM, and 4.47% by CRA method , whereas 38.55% are moderate biofilm producers by TCP method, 30.16% by TM and 1.68% by CRA method. In TM, 2 were found to be false positive and 23 false negative. In CRA method 3 were false positive and 89 false negative(Table/Fig 5),(Table/Fig 6),(Table/Fig 7).

Discussion

Bacterial biofilm has long been considered as a virulence factor contributing to infection associated with various medical devices and causing nosocomial infection (12),(13)

The exact process by which biofilm producing organisms cause disease is poorly understood. However, suggested mechanisms are:
i. Detachment of cells from medical device biofilm causing bloodstream or urinary tract infection.
ii. Endotoxin formation
iii. Resistance to host immune system
iv. Generation of resistance through plasmid exchange (2)

We isolated 179 Staphylococcal spp. from clinical samples, namely, blood, pus, urine, dialysis fluid, catheter, nasal swab etc. All isolates were isolated by standard procedure (15) and tested by three in vitro screening tests for biofilm production namely TCP, TM and CRA methods. Out of 179 Staphylococcal spp. 111 (62.01%) were S. epidermidis and 68 (37.99%) were S. aureus. We found that although the formation of biofilm on indwelling medical devices is generally associated with coagulase negative Staphylococci, S. aureus strains are also capable of production of biofilm (5.03%) which was observed by other workers also. (16),(17)

In this study antibiotic sensitivity pattern of various biofilm producers and non-producer Staphylococci spp. Isolated from clinical materials were obtained. The significant and clinically relevant observation was that the high resistance shown by biofilm producers to conventional antibiotics than non-biofilm producers. This observation was supported by other studies also (2),(10). All strains were sensitive to linezolid and vancomycin except two strains isolated from catheters which were intermediate vancomycin sensitive Staphylococcus aureus (VISA). Both were biofilm producers. Glycopeptides may not be optimal antimicrobial agents for the treatment of foreign body associated infection. This may be due to entrapment of vancomycin by the extracellular mucopolysaccharides because of their high molecular weight (10).

We adopted modified TCP method with extended incubation period for 24 hours instead of 18 hours. Trypticase soy broth with 1% glucose medium was used. This method was claimed superior to other methods by various researchers using Trypticase soy broth without glucose and Brain heart infusion broth with sucrose (11).

In TCP method biofilm formation was observed in 97 (54.19%) isolates and non-biofilm producers were 82 (45.81%). This study is similar to the observation made by Mathur et al (11). In tube test method, 76 (42.46%) isolates were found as biofilm producers whereas 103 (57.54%) were non-biofilm producers. In CRA, 11 (6.15%) strains produced biofilm and 168 (93.85%) were non-biofilm producers. Rate of positivity in CRA method in our study is higher than that of Mathur et al.

For data calculation, OD values obtained for individual strains of staphylococci spp.(11) mean OD values < 0.120 was considered non-biofilm producer, 0.120 – 0.240 was moderate and > 0.240 was considered as strong biofilm producers. Modified TCP method was considered as gold standard for this study as various researchers proved this method superior to standard TCP method using Trypticase soy broth without glucose. (8),(11)

Considering modified TCP as gold standard, data from TM and CRA methods were compared. Parameters like sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) were calculated. True biofilm producers were positive by modified TCP, TA and CRA. False positive were biofilm producers by TM and CRA method but not by modified TCP method. False negatives were non-biofilm producers by TM and CRA methods but the same strains were biofilm producers by modified TCP method. True negatives were non-biofilm producers by all the methods. In our study 3 strains gave false positive result and 89 false negative by CRA method. By TM only 2 strains were false positive and 23 false negative considering modified TCP method as gold standard.

Comparative analytical study of TM and CRA methods, with respect to modified TCP method which was considered as gold standard in this study, was as follows: Sensitivity of CRA method was 8.25%; specificity 96.34%; PPV 72.72%; and NPV 47.02%. Sensitivity of TM method was 76.27%; specificity 97.56%; PPV 97.36%; and NPV 77.66%.

Our study shows TCP is the better screening test for biofilm production than CRA and TM. The test is easy to perform and assess both qualitatively and quantitatively. In our study, positivity rate of CRA method was higher than observed by other workers, e.g. Mathur et al. Who has reported 5.26% biofilm producers by CRA method.

There are some highly accurate methods like PCR analysis to detect ica genes as virulence marker of staphylococcal infection. Biofilm non-producers are negative for icaA and icaD and lack the entire ica ADBC operon.[13,17] But in a developing country like ours, a low cost method for detection of biofilm is needed which require inexpensive equipment and less technical expertise.

Conclusion

Biofilm can be composed of a single or multiple organisms on various biotic and abiotic surfaces. There is association between biofilm production with persistent infection and antibiotic failure.(19) Hence, in infection caused by biofilm producing staphylococci, the differentiation with respect to its biofilm phenotype might help to modify the antibiotic therapy and to prevent infection related to biomedical devices. A suitable and reproducible method is necessary for screening of biofilm producers in any healthcare setup and this TCP method can be recommended.

References

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Donlan RM, Costerton W. Biofilms: Survival mechanisms of clinically relevant Microorganisms. Clinical microbiological review 2002; 15(2): 167-193.
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Carol A, Kumamoto, Marcelo DV. Alternative Candida albicans life style: Growth on surfaces. Annual Review of Microbiology 2005; 59:113-133.
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Murray EJ, Kiley BT, Stanley-wall RN. A Pivotal role for the response regulator DegU in controlling multicellular behavior. Microbiology 2009; 155: 1-8.
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Schwank S, Rajacic Z, Zimmerli W, Blaser J. Impact of bacterial biofilm formation on in vitro and in vivo activities of antibiotics. Antimicrobial agents and chemotherapy 1998; 42(4): 895-898.
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Crampton SE, Gerke C, Sehnell NF, Nicols WW, Gotz F. The intracellular adhesion (ica) locus present in staphylococcus aureus and is required for biofilm formation. Infection and Immunity 1999; 67(10):5427-5433.
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