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ANTIBIOFILM FORMATION AND WOUND HEALING ACTIVITY OF GLYCOPEPTIDE ANTIBIOTIC VANCOMYCIN AGAINST SKIN PATHOGENS

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Biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis were determined by the production of protease and lipase. Inhibition of lipolytic and proteolytic activity will lead to control of virulence factors produced by S. aureus and
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    Internationally indexed journal Internationally indexed journal Internationally indexed journal Internationally indexed journal Indexed in Chemical Abstract Services (USA), Index coppernicus, Ulrichs Directory of Periodicals, oo!le scholar, CA"I ,D#A$ , PS#A%, &"SC# , #pen $ !ate , Pro'uest , SC#PUS , &"AS& ,etc .   . Indexed in Elsevier Bibliographic Database (Scopus and EMBASE) SCImago Journal an! "#$%& Impact 'actor "#*   Rapid and Easy Publishing Rapid and Easy Publishing Rapid and Easy Publishing Rapid and Easy Publishing The “International Journal of Pharma and Bio Sienes! "IJPBS# is an international journal in English  published $uarterly. The aim of IJPBS is to publish peer re%ie&ed researh and re%ie& artiles rapidly &ithout delay in the de%eloping field of pharmaeutial and biologial sienes    +++#i,pbs#net *Instruction to Authors visit +++#i,pbs#net For any Queries, visit “contact” of www.ijpbs.net  Int J Pharm Bio Sci 2014 Jan; 5(1): (P) 232 - 248 This article can e !o"nloa!e! #rom """$i%&s$net P - 232 'esearch rticle Biotechnolo* International Jo+rnal o# Pharma an! Bio Sciences ISS, 0.5-/2   ,TIBII 'TI, , 6, 7I,9 TIIT<  9<PPTI ,TIBITI ,<I, 9I,ST S=I, PT79,S RANJITH KUMAR M* AND BRINDHA PRIYADARISINI V   Clinical Biotechnology Laboratory, Department of Microbial Biotechnology,  Bharathiar University, Coimbatore- 641046, n!ia" ABSTRACT Biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis  were determined by the production of protease and lipase. Inhibition of lipolytic and proteolytic activity will lead to control of virulence factors produced by S. aureus and S. epidermidis . Antibiofilm activity of isolated compound Vancomycin against S . aureus  and S. epidermidis  showed significant results. The effect of Vancomycin on Hydrophobicity Index HI!" dispersion on biofilm formation by microscopic method and glycocalyx production shows complete eradication of the biofilm formation. Vancomycin used in this study showed biofilm dispersion and disruption of the biofilm of the architecture by reducing #xopolysaccharide #$S! production and HI. The compound were studied for its wound healing property in rats and showed significant results compared to standard drug. The inhibitor used would have great clinical significance given to the difficulties encountered with treating S. aureus and  S. epidermidis  biofilm centered human infections. KEYWORDS: Biofilm formation, Vancomycin, wound healing, Skin infection  RANJITH KUMAR M  linical Biotechnolo* aorator*> e&artment o# icroial Biotechnolo*> Bharathiar 6ni?ersit*> oimatore- /4104/> In!ia$  Int J Pharm Bio Sci 2014 Jan; 5(1): (P) 232 - 248 This article can e !o"nloa!e! #rom """$i%&s$net P - 233 INTRODUCTION A biofilm is a sessile microbial community of cells that are attached to a substratum. These cells are embedded in a matrix of extracellular polymeric substances" and exhibit an altered phenotype with respect to bacterial physiology" metabolism and gene transcription % . &or example" genome'wide profiling of gene expression has shown that Staphylococcus aureus and  Staphylococcus epidermidis  biofilm is characteri(ed by a reduction in basic cell processes and induction of protective factors )* . +any pathogenic bacteria with the ability to form biofilms are responsible for acute and chronic infections. #xamples of biofilm'associated diseases are dental caries" gingivitis" periodontitis" endocarditis and prostatitis ,, . In addition" implanted medical devices including intravenous catheters" artificial -oints and cardiac pacemaers can become rapidly coated with human extracellular matrix and plasma proteins which are prime targets for bacterial biofilm formation %," %*" /* . Because of sharply decreased susceptibility of biofilm'forming bacteria to host defenses and antibiotic treatments" biofilms on implanted devices remain a ma-or medical problem %," /0" )) . There is presently no nown techni1ue that is able to successfully prevent or control the formation of unwanted biofilms without causing adverse side effects. In general" the formation of a biofilm involves several distinct stages" including initial attachment" cell'to'cell adhesion and proliferation" maturation" and finally" detachment /2 . Interestingly" in Staphylococcus " 3S appears to influence biofilm formation at many of these stages. Adhesion to a surface is the crucial transition stage from free'floating plantonic cells to a biofilm. This phenomenon involves the interplay of several adhesion molecules" which enable the physico'chemical interactions between the cells and the surface. 4on'functionality of the agr system facilitates the initial attachment of Staphylococci   to a polystyrene surface" presumably due to the up'regulation of attachment and down regulation of detachment molecules )2 . The surface'attached Atl# protein" a member of the Staphylococcal autolysin family" is over expressed in an agr mutant of S. epidermidis . This up'regulation of Atl# might modulate the adhesiveness of the bacterial cell surface. Since the identification of agr" it 1uicly became apparent that the agr system plays a central role in Staphylococcal pathogenesis. This present study mainly focuses on the antibiofim formation and wound healing property of Vancomycin against staphylococcus pathogens and used as biofilm inhibitors. An important stage in testing the potential of chemicals as antimicrobial drug candidates is establishment of their effectiveness in an animal model system %/" )5 . A useful animal model system should be clinically relevant" experimentally robust" ethically acceptable" and convenient to perform and should provide reliable and reproducible results. 6ound healing processes are well organi(ed biochemical and cellular events leading to the growth and regeneration of wounded tissue in a special manner. Healing of wounds involves the activity of an intricate networ of blood cells" cytoines" and growth factors which ultimately lead to the restoration to normal condition of the in-ured sin or tissue %5 . The process can be broadly categori(ed into three stages7 inflammatory phase consisting of the establishment of homeostasis and inflammation!7 proliferative phase consisting of granulation" contraction and epitheliali(ation! and finally the remodeling phase which ultimately determines the strength and appearance of the healed tissue ,0 . The aim of wound care is to promote wound healing in the shortest time possible" with minimal pain" discomfort" and scarring to the patient and must occur in a physiologic environment conducive to tissue repair and regeneration 2 . However" several clinical conditions and factors are nown to impair wound healing and these include hypoxia" infection" tumors" metabolic disorders such as diabetes mellitus" the presence of debris and necrotic tissue on the wound" certain drugs" and dietary deficiencies of protein" vitamins" or minerals )% . Topical antimicrobial therapy is one of the most important methods of wound care 88")8 . The goal of topical antimicrobial therapy in wound care is to control microbial  Int J Pharm Bio Sci 2014 Jan; 5(1): (P) 232 - 248 This article can e !o"nloa!e! #rom """$i%&s$net P - 234 coloni(ation and subse1uent proliferation thus promoting the healing of the wounds )8 . METHODS Strain maintenance S. aureus  and S. epidermidis  were procured from Institute of +edical Science" $S9 Hospitals" :oimbatore" Tamil 4adu" India and maintained in nutrient agar plates. Chemicals All the medium constituents and the antibiotic standards were procured from Himedia ;aboratories" +umbai" India. Solvents were purchased from &ischer Scientific" India. The compound Vancomycin was isolated in :linical Biotechnology laboratory" Bharathiar <niversity" :oimbatore" India.   Inhibition of protease Activity of S. aureus  and S. epidermidis was detected by using ;B agar plate. The strains were inoculated on ;B agar plates containing %= sim mil /5 . Single strea were made to the plate and protease producing colonies were detected by halo formation around the colony. Inhibition of proteolytic activity was detected by adding Vancomycin compound to the plate before streaing. ;B agar medium was used for 1ualitative analysis of extracellular proteinases. Small wells cut in the mil agar plates were filled with mixture of %5>g?m; of Vancomycin with )5>; ali1uots of cell free supernatants of S. aureus  and S. epidermidis  and of the respective bacterial supernatants. The inhibition of protease production was observed. Inhibition of lipase Tribuytrin agar medium ,*  was sterili(ed by autoclaving for ,5 min at %,%@:. The medium was shaen to form clear and transparent medium and pH was ad-usted to 0.5'0.8. +edium was poured and streaed with test organism and incubated at /0@:. If the microorganisms tested ha a lipolytic activity an opa1ue halo could be easily observed around the colonies. Inhibition of lipolytic activity were done by plate assay by adding )5>; of Vancomycin with cell free supernatants S. aureus  and S. epidermidis  to wells cut into the appropriate tribuytrin agar plates. The plates were incubated at /0@: for 8*h and evaluated by measuring (one of clearance. Cell Surface Hydrophobicity The effect on :SH of S. aureus  and S. epidermidis  were measured by bacterial adhesion to hydrocarbon BATH!  2% . Briefly" the bacterial cells grown in TSB broth were resuspended to an  of %.5C5.5% at 255 nm. The effect of Vancomycin on hydrophobicity index was measured by adding the Vancomycin to bacterial cultures and % m; of hexadecane was added to 8m; of ad-usted cell suspension in a test tube and was vortexed for % min. The mixture was allowed to settle and separate for /5 min" and the  of a1ueous phase was measured. The hydrophobicity index HI! of microbial cells was calculated by the formula described by Serebryaova et al  . 8* . The results were expressed as the proportion of the cells which were excluded from the a1ueous phase" determined by the e1uation D initial '  finalE ? D initial x %55E. Biofilm formation by microscopic method Biofilm formation was done according to the method of ;embe et al  . ,F . 9lass test tubes containing Tryptone soybean broth TSB! were supplemented with bacterial suspensions. Biofilm were allowed to grow on glass pieces in the glass test tubes supplemented with the %5>g?m; of Vancomycin compound as well as without the compound. The tube supplemented with the %= +S was taen as control and was incubated at /0@: for ,8h. After incubation" the glass pieces were washed four times with 5.*)= normal saline solution 4SS! and each tube stained was with 5.%= crystal violet solution for %5 min. Stained glass pieces were observed at a magnification of 85G using a ;ynx light microscope. Quantification method S. aureus  and S. epidermidis were grown in TSB medium supplemented with 5.,)= glucose and the biofilm formation was 1uantified spectrometrically at 8%)nm. The effect of the Vancomycin compound on biofilm formation was  Int J Pharm Bio Sci 2014 Jan; 5(1): (P) 232 - 248 This article can e !o"nloa!e! #rom """$i%&s$net P - 235 investigated by adding the compound into glass test tubes containing the medium supplemented with the bacterial suspension. The tubes containing the media and the compound were taen as control.  Adherent assay: Tube method 9lycocalyx production was determined as described by :hristensen et al  . * . S. aureus  and S. epidermidis  were inoculated into )m; of TSB in glass tubes in triplicate. Saccharide free basal medium TSB without glucose! that lacs the substrate for polysaccharide was used as a control. :ulture was incubated at /0@: for ,5',8h and the contents were aspirated. ne tube was examined unstained and one each stained with tryptan blue and safranin. Slime positivity was -udged by the presence of visible unstained or stained film lining the wall of the tube. &ormation of a ring at the li1uid air interface was not considered as positive test. Inhibition of glycocalyx production was confirmed by slime negativity. The tubes were also examined by phase contrast microscopy to detect the presence of adherent microcolonies. Effect of antibiotic compound against bacterial growth cure   The effect of Vancomycin compound on cell proliferation of S. aureus  and S. epidermidis  were determined. Briefly" an overnight culture TSB medium! of S. aureus  and S. epidermidis  were diluted to obtain 5.%  at 255nm. Anti'biofilm activity and the effect of growth were regularly monitored at ,h intervals until a final time point of ,8h.  Animal trail Healthy 6istar rats of either sex and of approximately the same age" weighing about %)5',)5 g were used for the study. The animals were maintained in the animal house in the epartment of $harmacology" +:H Health Sciences and esearch Institute. They were divided into nine groups n J 2! for the studies. ats were allowed to acclimati(e in the research laboratory for 8 days before the commencement of the study and were fed with standard livestoc pellets $ranav agro industries ;td." Saangli" +aharashtra!. The animals were allowed for unrestricted access to clean drining water. The experimental protocols were sub-ected to scrutiny of Institutional Animal #thics :ommittee for experimental clearance +:#T?$h.?%/?,5%%!. !ormulation of the ointment Two different doses of the ointments were prepared and used in the study. 9roup /" 8" 0 and * obtained two different concentration of the Vancomycin compound %= and ,= of the ointment base!. 9roup F were treated with 4eomycin standard. The re1uired 1uantities of antibiotics was weighed" added to the molten ointment base and then homogeni(ed by trituration method as mentioned in British $harmacopeia ,  was followed for the preparation of ointment. The hard paraffin 5.) g! and cetostearyl alcohol 5.) g! were melted on the water bath. 6ool fat 5.) g! and yellow soft paraffin *.) g! were incorporated in to it" stirred until all the ingredients were melted. &oreign particles were removed by decantation and the mixture was stirred thoroughly until cold. The Vancomycin powder %55 mg for %= and ,55mg for ,=! was triturated with the above simple ointment base on an ointment slab.  Acute oral to"icity studies Acute oral toxicity was performed as per organi(ation for economic co'operation for development #:! guideline 8,/ methods. The Vancomycin powder was administrated in a single dose by gavage using specially designed mice oral needle. Animals were fasted / h prior to dosing food was withheld for / h but not water!. &ollowing the period of fasting animals was weighed and test substance was administrated orally at a dose of %55" )55" %555 and ,555 mg? g. After administration" food was withheld for ,h in mice. Animals were observed individually at least once during the first /5 minutes" later periodically during the first ,8 hrs" with special attention given to the first 8 hrs" and daily thereafter" for a total of %8 days. #ound healing studies 6istar rats of either sex were used for this study. A round seal of %) mm diameter was impressed on the sides of the central trun depilated and
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