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

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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 Dermatolgy,
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
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,
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,
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
(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)
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.
On April 2011

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.

Dr. Anuradha
On Jan 2020

Important Notice

Year : 2011 | Month : February | Volume : 5 | Issue : 1 | Page : 127 - 130 Full Version

Biofilms: A Challenge To Medical Science

Published: February 1, 2011 | DOI:

*(M.D.) Professor of Microbiology **(M.D.) Professor of Medicine Rural Medical College, Loni, Ahmednagar (M.S.) India

Correspondence Address :
Dr. Seema Bose
Professor of Microbiology
Rural Medical College
Ahmednagar, 413736 (M.S.) India


Biofilms are a group of microbes which are encased in an exopolysaccharide matrix on both biotic and abiotic surfaces. Various changes occur during their transition from a planktonic to a surface attached community. This causes a number of persistent infections which respond poorly to conventional antibiotic therapy. This review summarizes the nature, prevalence, detection and the treatment of biofilm associated infections.


Biofilms, Indwelling medical devices, Tissue culture plates, Multi drug resistant

The first recorded observation concerning biofilms was probably given by Henrici in 1933, who observed that water bacteria were not free floating, but that they grew on submerged surfaces.(1)
A biofilm is a sessile community of microorganisms which are attached to an interface or to each other and are embedded in an exopolysaccharide matrix. It manifests an altered growth rate and transcribes genes that free floating organisms do not transcribe.(2) After adherence to a surface, these microorganisms adapt to the environment of the biofilm by increasing the secretion of exopolysaccharide. This helps the microorganisms to escape their killing by antibiotics.(1)
The mechanism of biofilm formation
There are five steps of biofilm formation on medical devices.(3)
In steps 1 and 2, the identification and association with a surface is followed by strong adhesion. The time taken by this is 1 to 2 hours post – implantation. These reversible, non specific cellular associations occur through long

and short range forces, e.g., van der Waal’s forces, gravitational forces, hydrogen bonds, hydrophobic interactions, etc.
In steps 3 and 4, microbial cells aggregate to form micro colonies. Thereafter, further growth and maturation of the biofilm takes place in the next 2 – 3 hours. Specific chemical reactions between the compound of the microbial cells and the substrate surfaces result in strong adhesion and irreversible molecular bridging. The biofilm which is formed can be of a flat or mushroom shape, which depends upon the nutrient source. Microbial polysaccharide and adhesin proteins promote the attachment of organisms to the substrate surfaces.
In step 5, sloughing of the biofilm into small pieces occurs and these pieces move transiently to form daughter cells. The daughter cells which are thus formed, travel down through the blood stream to various new attachment sites.
Transitions to a sessile state of bacteria occur in response to the limitation of essential nutrients. Biofilm formation is commonly regulated by inter and intraspecies quorumsensing mechanisms. Availability of nutrients, chemotaxis towards the surface, the motility of bacteria, surface adhesion and the presence of surfactants, influence biofilm formation in microorganisms.(4)
By using bacillus subtilis, Dr Stanley Wall has shown that a protein called Deg U regulates biofilm formation.(4)
Certain surface proteins, extracellular proteins, capsular polysaccharides and adhesins PS/A and autolysin (encoded by the atl E gene) regulate biofilm production. The ica genes also code for PS/A and intracellular adhesion.(5)
Biofilms may be formed by one or several types of microorganisms. Studies on polymicrobial biofilms which are formed by Candida albicans and Staphylococcus epidermidis indicate that biofilms which are produced, may protect the fungus from antifungal action.(6)
Many researchers have shown that the regulation of ica operon and the formation of biofilms depend upon various environmental factors like anaerobicity, carbon dioxide level and glucose and osmotic levels. Sodium chloride is a known activator of ica operon transcription. Some workers have found that sodium chloride mostly induced biofilm formation among methicillin sensitive staphylococcus aureus. They also found that biofilm production among methicillin resistant staphylococcus aureus was mainly glucose induced.(7)

There was a variable adherence of the microorganisms to the polystyrene surfaces in vitro. This may be due to the variation in different strains and due to the expression of the genes which are responsible for biofilm production. Another hypothesis is that, in the inserted catheter under in vivo conditions, several host proteins coat the catheter surface. The microorganisms lodge to the coat by using multiple receptors.(7), (8) The various factors such as surface area, the type of surface (rough/smooth), porosity, charge on the surface and surface hydrophobicity play a role in the formation of the biofilm. A rough surface is more favourable for the colonization of bacteria. The hydrophobicity in polymeric materials increases biofilm formation. Microorganisms get attached easily on porous surfaces. Electrostatic interactions cause biofilm cohesion. Cations contribute to the cross linking of the biofilm matrix.(3)
The extent of initial adhesion depends upon the adherence property of the receptacle, the duration and the number of bacteria coming in contact with the test surfaces and the fluid turbulence of the test media.(9), (10)
Exopolysaccharides are formed under selective pressure and are controlled by diffusible chemical signals (quorum sensing) of the cells within the biofilm. So, the biofilm is not homogeneous.(3), (11)
A study showed that the addition of pheromones to biofilm forming Enterococcus faecalis yielded a high amount of biofilm formation.(12)
Another group of workers demonstrated a strong association between the biofilms which were produced by the clinical isolates of Acinetobacter baumanni and multiple drug resistance. The presence of extended spectrum beta lactamases (bla PER1) is likely to facilitate cell adherence.(13)
The prevalence and the expression of F-like conjugative pili, adherence fimbri and curly, which are known to promote biofilm formation in Escherichia coli K12, cannot totally account for the increased biofilm formation of non domesticated Escherichia coli in vitro.(14)
Biofilm associated infections and their implications in health care
According to a recent public announcement from the National Institutes of Health, more than 60% of all the infections are caused by biofilms.(15) As described by Prasanna et al, about 40-50% of adults had biofilm related gingival infections. Among 4000 infants with cerebrospinal- fluid shunts, 15-20% had biofilm related infections. 95% of the urinary tract infections were associated with urinary catheters. 86% pneumonias were associated with mechanical ventilation and 85% of the blood stream infections were closely related to intravascular devices.(3)
The suggested roles of the biofilms in producing infections are –
a. Detachment of the cells – the cells may get detached from the biofilm. This may cause blood stream and urinary tract infections.(16)
b. Resistance to the host immune system – Biofilm coated bacteria escape the damaging effect of the antibodies produced by the infected host cells.(17)
c. Production of endotoxins – Gram negative bacteria which are encased in biofilms, produce endotoxin.(18)
d. The generation of resistant organisms – Bacteria can transfer plasmids by conjugation within the biofilm. So, resistance factors may be exchanged through a plasmid.(19)
Two types of biofilm associated infections can occur –
1. Foreign body infections
2. Native tissue infections
Foreign body infections – These are more commonly associated with the colonization of microbes on indwelling medical devices (IMD). IMDs may cause the haematogenous spread of infections throughout life if the devices are in place.
For surgical IMDs, tissue damage and clot formation are associated with surgical implantation, thus causing increased microbial biofilm formation.
For non surgical IMDs, e.g. Urinary catheters, colonization may occur from skin or through or around catheters, once they are implanted.
Native tissue infections – Some biofilm related infections involve no foreign bodies eg. Urinary tract infections by uropathogenic Escherichia coli, cystic fibrosis by Pseudomonas aeruginosa, native valve endocarditis by streptococcus viridians, etc.(1),(2)
Mechanisms of antimicrobial resistance of biofilms
Microbial biofilms have been associated with a variety of persistent infections which respond poorly to conventional antibiotic therapy. This also helps in the spread of antibiotic resistant traits in nosocomial pathogens by increasing mutation rates and by the exchange of genes which are responsible for antibiotic resistance. Antibiotic therapy against device associated biofilm organisms often fails without the removal of the infected implant. An elevated expression of the efflux pump is another mechanism for the development of antibiotic resistance in biofilm bacteria. The specific up regulation of genes which encode antibiotic transporters, has been seen in biofilms which are formed by Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Physiological heterogeneity is another important characteristic which is observed in biofilm bacteria. This phenomenon affects the rate of growth and metabolism of the bacteria and is reflected by interbacterial quorum signals, the accumulation of toxic products and the change in the local micro environment. These so called persister cells are not resistant to antibiotics per se, but become resistant when associated with the biofilm.(9)
The overall healthcare mechanisms of the underlying antimicrobial resistance of biofilms are:
1. Trapping of antibiotics in the exopolysaccharide matrix – The exo polysaccharide slime causes a diffusion barrier by restricting the rate of molecule transport to the interior of the biofilm, or by chemically reacting with the molecules themselves. The exopolysaccharide which is negatively charged, restricts the penetration of the positively charged molecules of antibiotics by chemical interactions or by molecular binding. This also dilutes the concentration of the antibiotics before they reach to the individual bacterial cells in the biofilm, thus making the antibiotics less effective against microorganisms.(1), (2)
2. Bacteria which are coated with biofilms escape the host immune system – Biofilm bacteria escape the damaging effect of the antibodies which are produced by the host immune system in response to infections.(16)
3. Quorum sensing and genotyping adaptations alter the metabolism and decrease the growth rate of bacteria- A cell to cell communication in bacterial biofilms is established through chemical signaling. Small, diffusible molecules of class of N – acylated homoserine lactones (AHLs) are liberated by biofilm bacteria into their surrounding environment. These AHLs are associated with DNA binding proteins. As the amount of AHLsreaches a threshold level, it induces the transcription of specific genes throughout the population. The regulation of this type is known as quorum sensing (Requirement of a specific population of bacteria that is nessesary for the activation of the AHL – responsive genes). The cells lying deep within the biofilm have less metabolic activity and growth rates. This makes the biofilm organisms inherently less susceptible to antibiotics. Due to the consumption of oxygen and glucose, relative anaerobiasis is created at the deeper layers of the biofilm, where in order to survive, the microorganisms transform into slow growers and non growers. Older biofilms are relatively more resistant than newer biofilms.(3)
After the attachment to a biotic or an abiotic surface, the bacteria undergo further adaptation, i.e, increased synthesis of exopolysaccharide and increased antibiotic resistance. They also develop an increased resistance to UV light, increased genetic exchange, altered metabolism and increased secondary metabolic production.(1), (2)
The detection of biofilm producing microorganisms
Early biofilm formation detection might result in a greater success in the treatment, because in long standing cases, they may be very damaging and may produce immune complex sequelae.(2) There are two methods for the detection of biofilms –
1. The Phenotypic method
a. The tissue culture plate (TCP) method – The wells of the tissue culture plates are inoculated with a bacterial suspension along with positive and negative controls and these are incubated for 24 to 48 hours. Planktonic cells are removed by washing with phosphate buffered saline. Biofilms are fixed with 2% sodium acetate and are stained with 0.1% crystal violet. The excess dye is washed away with deionised water. The plates are dried properly and the optical densities of the stained biofilms are obtained spectrophotometrically.
b. The tube method(TM) – 10 ml of Tripticase soy broth with 1% glucose is inoculated with a loopful of test organisms, along with positive and negative controls. The broths are incubated at 370C for 24 – 48 hours. The culture supernatants are decanted and the tubes are washed with phosphate buffered saline. The tubes are dried and are stained with 0.1% crystal violet. The excess stain is washed away with deionised water. The tubes are dried in an inverted position.
c. The Congo red agar (CRA) method – The Congo red stain is prepared as a concentrated aqueous solution and is autoclaved at 1210c for 15 minutes. This is added to autoclaved Brain heart infusion agar with sucrose at 550c. The plates are inoculated with the test organisms along with positive and negative controls and are incubated at 370C for 24 to 48 hours aerobically. Black colonies with a dry crystalline consistency indicate biofilm production.
Various studies have established that TCP is a better screening test for biofilm production than the TM and the CRA methods. The test is easy to perform and to assess biofilms, both qualitatively and quantitatively. (20), (21)
2. The Genotypic method
Sonications and PCR amplification methods have been shown to improve the detection of biofilms. Biofilm non producers are negative for ica A and ica D and lack the entire ica ADBC operon. But this requires specialized equipments and techniques.(22), (23)
Prophylaxis against biofilm formation
This includes systemic perioperative and local antibiotic prophylaxis. The aim of the local antibiotic prophylaxis is to inhibit the colonization of microorganisms on devices and the contamination of the surrounding tissues. Antimicrobials can be applied locally in various forms, such as: -
1. Device coating – Devices are coated with antibiotics or quorum sensing inhibitors, which are either covalently bound to the device, or are locally eluted from it. Device coatings are of two types – active and passive. Passive coating such as ethylene glycol, poly ethylene oxide and hydrophilic poly urethane can be used. The effectiveness of passive coating is limited. In active coating, the release of anti microbial agents in high fluxes occur to inhibit the initial adhesion of bacteria.(3), (9), (24), (25)
2. Device immersion – The dipping of the device in antimicrobial solution, e.g., rifampicin dipped vascular graft.(25)
3. Surgical site irrigation – Skin antisepsis and the antimicrobial irrigation of the surgical field.(25)
4. Antibiotic loaded cements – The use of antibiotic loaded bone cements (usually in joint arthroplasties) provides the local delivery of antibiotics, the stabilization of soft tissues, scope for an easier re implantation and better clinical outcome.(26)
5. Antibiotic lock therapy – The catheter lumen is filled with the concentrated antibiotic solution and is then β€œlocked” into place for an extended period, when not in use. This is done to prevent the colonization by bacteria.(9)
6. Antimicrobial carrier – Antimicrobials can be added onto a carrier either preoperatively or during surgery. Biodegradable (polylactide or polygalactide) or non biodegradable (poly methyl methacrylate) polymers which are impregnated with antimicrobials are used in orthopaedic prostheses. The resulting effect of the antimicrobials persist for weeks to months.(25)
Antibiotic prophylaxis is controversial, but is increasingly common in the high risk patient group.(9)
The common treatment against persistent infections which are produced by biofilm producers is the removal of an infected IMD, combined with antibiotic/antifungal therapy.
In case of IMD in non surgical patients, long term antibiotic therapy is required.(24), (27), (28)
Experimental therapy
1. The in vitro use of ultrasound electric fields to enhance the penetration of antibiotics through microbial biofilms – Devices, which emit low energy surface acoustic waves, electric currents, or pulsed ultrasound reduce the colonization of the devices and enhance the release of locally applied antibiotics.(3), (9)
2. Proteolytic enzyme treatment, e.g, alginate lysate in case of the polysaccharide biofilm of Pseudomonas aeruginosa.(1)
3. The evaluation of newer antibiotics and microbicide immersion practices. (3)
4. The disruption of signaling molecules (acylhomoserine lactones) acting as quorum sensing systems. These are involved in the biofilm architecture and detachment, e.g. palulin and penicillin acid, which are secondary metabolic products of the Penicillium species, act as quorum sensing inhibitors.(29)
5. Inhibition of biofilms by small molecules – Designing small molecules which can prevent biofilm formation at some specific point. Amino imidazole, triazole and tether units together form a conjugate which can disperse bacterial biofilms without causing bacterial death. Short carbon chain molecules (decanol, decanoic acid and dodecanol) can inhibit and disrupt biofilms in a concentration dependent manner.(3), (30)
6. In the future, the treatment that inhibits the transcription of the biofilm regulatory genes might be able to completely inhibit biofilms. Identifying the virulent factor and genes which cause biofilm formation, can help in preventing the colonization of the microorganisms.(1), (9)
7. Use of sensors – The inhibition of biofilms after their complete formation is difficult because of the presence of β€˜persister’cells. Sensors which can detect biofilm formation as early as possible, are a great help for the treating clinicians. Research is going on to make several types of sensors for biofilm monitoring, such as bacterial touch sensors and electro chemical sensors (non bacterial sensors). Overexpression of VPsS, a hybrid sensor kinase, enhances biofilm formation in Vibrio cholerae (bacterial sensor).(31)
8. Chitosan, a polymer which is isolated from the crustacean exoskeleton, inhibits candidal biofilm formation in vivo. It damages the fungal cells. Therefore, it can be considered for the prevention or the treatment of the fungal biofilms of the central venous catheters and other medical devices.(32)


Many biofilm infections develop slowly, producing very few symptoms initially, but in the long run, they may produce immune complex sequelae and may act as reservoirs of infection.(2)
Standard, in vitro antibiotic susceptibility tests are not predictive of the therapeutic outcome of biofilm associated infections. (28) The overall healthcare costs which are attributed to the treatment of biofilm associated infections are much higher due to their persistence. Besides, a longer hospital stay is another factor for higher costs. Early detection of biofilm associated infections and newer treatment options for the management of the same are needed.


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