International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
35
Effect of chitosan coated chemogenic silver nanoparticles coated syringes
against biofilm of clinical isolate of Staphylococcus aureus
S.Karthick Raja Namasivayam, Pawan Kumar, S.Kiran Nivedh, A.N.Nishanth, EAllen
Roy
Department of Biotechnology, Sathyabama University, Chennai 119, Tamil Nadu, India
*Corresponding author e-mail address biologiask@gmail.com
ABSTRACT
Biofilm represents the most prevalent type of virulent factor of most of the pathogenic microorganism
and involved in crucial development of clinical infection and exhibit resistance to antimicrobial agents.Now the
biofilm is considered as major target for the pharmacological development of drugs. A biofilm serves to
promote bacterial persistence by resisting antibiotic treatment and host immune responses. Antibiotics are
rendered ineffective when biofilms form due to their relative impermeability, the variable physiological status of
microorganisms, subpopulations of persistent strains, and variations of phenotypes present .Metal
nanotechnology chemistry has the potential to prevent the formation of these life-threatening biofilms on life
supporting devices.In the present study, anti biofilm effect of silver nanoparticles coated syringes against
clinical isolate of Staphylococcus aureus was studied. Chitosan stabilized silver nanoparticles synthesized by
IJOART
chemical reduction method and the synthesized particles were coated on the surface by ultrasonication. Coated
syringes were characterized by scanning electron microscopy (SEM) which reveals complete dispersion of the
nanoparticles on the fibre surface and the size, shape of the particles shows uniform spherical particles with the
size of 60-70 nm. Distinct effect of biofilm inhibition was recorded in the nanoparticles coated syringes and
maximum inhibition was observed during 72 hour of incubation. Biochemical composition of biofilm matrix
mainly total carbohydrates and total protein was highly reduced. The present study would suggests the
development of anti microbial coated medical devices against pathogenic microorganism.
Keywords. Biofilm, Staphylococcus aureus,Silver nanoparticles,Chitosan
1.INTRODUCTION
Nanobiotechnology, the convergence of nanotechnology and biotechnology and in particular
its applications in the medical sector are considered as one of the most promising and most advanced
areas of nano technology[1]. The application of nanotechnology in the field of healthcare has come
under great attention in recent times. There are many treatments today that take a lot of time and are
also very expensive. Using nanotechnology, quicker and much cheaper treatments can be developed.
By performing further research on this technology, cures can be found for diseases that have no cure
today. The application of such a technology can be used for the inhibition of biofilm formation on the
surgical and medical devices which are of higher threat in the process of treatments. Bacteria are able
to grow adhered to almost any surface, forming architecturally complex communities termed biofilms
[2,3]. Microbial biofilms develop when microorganisms irreversibly adhere to a submerged surface
and produce extracellular polymers that facilitate adhesion and provide a structural matrix. This
surface may be inert, nonliving material or living tissue. Biofilm-associated microorganisms behave
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
36
differently from freely suspended organisms with respect to growth rates and ability to resist
antimicrobial treatments and therefore pose a public health problem [4, 5]. Due to increasing tolerance
of the biofilm community to antibiotics, biocides and mechanical stress, it has become just as difficult
to completely eradicate mature biofilms as it is to completely avoid the presence of planktonic cells,
the origin of the biofilm in the water. Common treatments to prevent or remove biofouling include
using disinfection, minimizing nutrients in the feed or altering surface materials to prevent bacterial
attachment, or clean-in-place (CIP) to remove mature biofilm by chemical or mechanical shear.
Several studies have examined the effect of various types of antimicrobial treatment in controlling
biofilm formation on medical devices[6, 7, 8]. The vast majority of the chemical agents currently
available for biofilm control are broad-spectrum non-specific micro biocide agents [9]. Chloro
hexidine, triclosan, and essential oils (e.g., Listerine) are the most commonly used and clinically tested
antimicrobials [10]. Biofilm-control strategies based on disruption of EPS formation on the surface
could be an effective alternative (or adjunctive) approach [11]. In order to control biofilm formation
on medical devices and all costs associated, a large number of new strategies and approaches have
been developed in the last few years, including: antimicrobial locks (in the case of catheters) [12];
surface modification of biomaterials with antimicrobial coatings [13]; the use of quorum sensing (QS)
IJOART
inhibitors [14], antimicrobial peptides as a new class of antibiotics [15]; enzymes that dissolve
biofilms [16], nitric oxide [17], electrical [18] or ultrasound [19] enhancement of antimicrobial
activity, or even the application of light activated antimicrobial agents [20]. Nevertheless, nanoscale
materials have recently appeared as one of the most promising strategies to control biofilm infections
related to indwelling medical devices, especially due to their high surface area to volume ratio and
unique chemical and physical properties [21]. A nanomaterial has a diameter ranging from1 and 100
nm, and they can be made from different materials, like copper, zinc, titanium, magnesium, gold,
alginate and silver. The use of silver nanoparticles (NPs) is now considered as one of the most
promising strategies to combat biofilm infections related to indwelling medical devices [22]. Drug
delivery nano carriers systems, such as liposomes [23] and polymer-based [24] carriers have also
arisen as appealing methods with a great potential in the treatment of biofilm infections, due to several
factors especially good biocompatibility and ample range and extent of drugs that they can carry.
Another important factor is the protection provided by the encapsulation of the drug in the biological
milieu, decreasing toxicity and allowing the drug to reach the specific site [25]. Chitosan is another
natural polymer has been reported as a polymer-based protective agent to stabilize the metal
nanoparticles[26].Because of the biocompatibility,biodegradability, nontoxicity and adsorption
properties of chitosan, it was used as a stabilizing agent to prepare Ag, Au and Pt nanoparticles.
These chitosan- protected nanoparticles can be easily integrated into systems relevant for
pharmaceutical, biomedical, and biosensor applications. Therefore, it has attracted considerable
interest due to its medicinal properties, such as antifungal, antibacterial, antiprotozoal, anticancer,
antiplaque, antitartar, hemostatic, wound healing and potentiates anti-inflammatory response, inhibits
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
37
the growth of cariogenic bacteria, immunopotentiation, antihypertensive, serum cholesterol lowering,
immune enhancer, increases salivary secretion (anti-xerostomial) and helps in the formation of bone
substitute materials[27].The present study is aimed to evaluate anti biofilm effect of biocompatible
polymer stabilized metallic nanoparticles coated syringes against clinical isolate of Staaph. aureus
under in vitro condition.
2.MATERIALS AND METHODS
Coating of metallic nanoparticle on syringes
Syringes were obtained from Solaguard (Chennai,Tamil Nadu,India). The outer transparent portion of
syringes were cut into 4 pieces and transferred to beaker containing 0.1 molar AgNO 3 and 0.1molar tri
sodium citrate placed in ultrasonicator. Freshly prepared 0.1 molar sodium borohydride was added
drop by drop till reaction mixture turned into brown. The preparation was left in ultrasonicator for 2
hours to facilitate complete dispersion of nanoparticle on surface. The coating of nanoparticles on
syringe was primarily confirmed by colour change of cut pieces of syringe into brown colour. The
pieces were dried at 40oC overnight to remove excess moisture. The dried pieces were kept in sterile
petriplate for further study. Chitosan coated nanoparticle was also coated by chemical reduction
method of respective metal precursor with reducing agent and 0.1 molar chitosan as a stabilizer agent.
IJOART
Chitosan was obtained from SRL laboratory and deacetylation process was done and degree of
deacetylation was determined using Viscometric method. The pre-treated chitosan as described earlier
was dissolved in 1% w/v acetic acid (1 mL of acetic acid in 100 mL of distilled water) and suspension
was transferred to a beaker containing respective metal precursor and a reducer. The homogenous
slurry thus obtained was coated with cut pieces. Before coating, the suspension was characterized by
scanning electron microscopy (SEM equipped with energy dispersive x ray atomic spectroscopy
(EDAX),
the mixtureThe coated cut pieces thus obtained were dried at 40oC as described earlier.
Biofilm inhibition assay
The metallic nanoparticles coated syringes kept in sterile Petri plates were inoculated with 5 mL of
S.aureus culture. The plates were allowed for incubation at 37oC for 72 hours. After incubation period
the treated syringes were stained with 0.1% w/v of crystal violet solution for 15 minutes at room
temperature. After staining the syringe pieces were washed with phosphate buffered saline (PBS)
solution to remove free planktonic cell. Further washing was carried out with 95% of ethanol for 3
times at room temperature and the washed solution was collected and absorbance was measured
spectrophotometrically at 540nm.
The percentage of biofilm inhibition was calculated by following formula:
Biofilm inhibition (%) = OD in control – OD in treatment × 100
OD in control
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
38
Biofilm Kinetics
Biofilm kinetics was done to study the inhibition percentage of nanoparticles coated syringes
against biofilm of Staphylococcus aureus with respect to time. Fresh syringe was taken and dispersed
in metallic nanoparticles - AgNp, AgNp-CS. The syringe was kept in beaker containing AgNp and
kept for sonication in water bath sonicator for 3 hours likewise performed for AgNp-CS and kept them
in water bath sonicator for 3 hours. Once coating was done, the Petri plates were kept in dry air oven
at 46oC for 2 hours. After drying, inoculation was done by spraying 5 mL of Staphylococcus aureus on
different nanoparticle coated syringes and kept for incubation for different time interval. Fresh syringe
was taken as control in another petri plate. After 12 hours of incubation, Ag coated syringe was cut
into 1st part with surgical blade and the remaining part was kept for further incubation. Similarly
AgNp-CS coated syringe was cut and the remaining portion was kept for incubation. Incubated coated
syringes and control syringes were dipped in 2 mL of 0.1% w/v of crystal violet in sterile boiling test
tubes each, shaken properly and kept for incubation at room temperature for 15 minutes. After the
incubation period crystal violet was removed with sterile micro tip then the syringes were washed with
2 mL of sterile phosphate buffered solution twice. It was aspirated and PBS was discarded, 5 mL of
IJOART
ethanol was added to each tube and was kept on ultrasonicator for 15 minutes. Elutants were measured
at 540nm spectrophotometrically and reading was kept for tabulation.
Evaluation of effect of nanoparticles on biochemical composition of biofilm matrix
Biochemical composition of biofilm matrix mainly total carbohydrate and total protein was
carried out. The control syringe pieces and respective nanoparticle coated syringe pieces (3 in each
treatment) was transferred to test tube each containing 5 mL of culture containing S.aureus prepared
overnight. Test tubes containing pieces and culture were kept for incubation at 37oC for 3 days for
allowing formation of biofilm on the syringes
After incubation period, the inoculated pieces were transferred to screw cap vials containing 5
mL of 0.9% NaCl. The bottles were sonicated for 10 minute in an ultrasonicator water bath and
vortexed vigorously for 1 minute to disturb biofilm. Cell suspensions were then folded and centrifuged
at 10000 rpm at 4oC for 10 minutes.The collected suspension was used as source for studying
biochemical composition in terms of total protein determined by Lowry et al and total carbohydrate by
Anthrone method.
3.RESULT AND DISCUSSION
Chitosan stabilized silver nanoparticles were synthesized by chemical reduction of metal salt
precursor with
nontoxic and biocompatible polymer chitosan
FTIR.SEM and EDAX. When the FTIR spectrum of
which primarily confirmed by
free and stabilized
nanoparticles were
compared, it was found that almost the all the absorbed peaks were modified upon coating with
chitosan. FTIR spectra of chitosan coated silver nanoparticles are presented in (Figure 1 a,b).The IR
spectra of the chitosan capped Nano silver shows prominent peaks at ≅ 3788 cm-1, 3427.4005 cm-1
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
39
corresponding to O – H stretching, strong polymerization, at 2928.1733 cm-1 for aliphatic C – H
stretching. Peaks at approximately 2369.0387 cm-1,2345.3187 cm-1 represent N – H stretching
vibration. Peaks at 1637.5845 cm-1 and 1389.4649 cm-1 represent N –H bending and a peak at
1026.6957 represents C – N vibration in aliphatic compounds. A is also observed at 617.7611 cm-1
showing the presence of inorganic metal ions (silver ions). SEM analyzer built in with and EDAX
analyzer allows a quantitative deduction on localization of elements in the nano specimens Scanning
electron microscopy (SEM) study of chitosan stabilized silver nanoparticles reveals. the uniform
spherical smooth morphology. within the size range of 101.78 nanometers and electron dense thin
chitosan coating shell of diameter 3-5 nanometers(Figure2 a) Such size distribution analysis primarily
confirms that the particles are well dispersed and less aggregated The EDAX images illustrated the
presence of large amounts of C, O, N (Figure 2 b).
Coating of respective nanoparticles on syringes by ultrasonicator was primarily confirmed by
fine dispersion of particles on the surface which can be easily visualized. Surface topography with
SEM clearly reveals uniform spherical particles with nano range embedded on the syringe surface
(Fig. 3.) SEM Micrograph reveals complete disturbance of biofilm, less aggregates, weakened cell
mass was observed (Figure 3a). Biofilm inhibition study clearly reveals all the nanoparticles (both free
IJOART
and coated) inhibited biofilm in significant manner (P.0.05). Maximum inhibition of 80.4% was
recorded in AgNp-CS treatment. followed by. 72.8% was reported from free Ag and (Table 1).
Biofilm kinetics
Biofilm kinetics study clearly reveals all the tested nanoparticles inhibited biofilm with
respect to different time interval ranging from 12, 24, 36, 48, 60, 72 hours, but distinct effect was
observed in chitosan coated nanoparticle and linear increase in inhibitory effect was inferred during
late inhibition period.
In the case of free AgNps the biofilm inhibition at respective time period was found to be 9.8,
11.7, 15.9, 21.05, 34.3, 41.6 % (Table .2.). Improved inhibitory activity was reported from CS coated
AgNp as 11.2, 14.6, 19.3, 26.1, 40.7, 49.8 % during 12, 24, 36, 48, 60, 72 hours respectively (Table
3.)
Effect of Nanoparticles on biochemical composition of biofilm matrix of S.aureus
All the tested nanoparticles (both free and coated) reduced biochemical composition mainly
total protein and total carbohydrate of biofilm matrix (Table 4). Maximum reduction of protein was
recorded in chitosan coated syringes (12 mg/mL) followed by AgNp-CS (15 mg/mL). free Ag
nanoparticle recorded was (17 mg/mL).Similar effects on carbohydrate content was also reported
from CS and AgNp-CS nanoparticles which recorded least total carbohydrate content (14 mg/mL)
each followed by AgNp (16 mg/mL).
The developments of nanoparticles with antimicrobial properties have recently
received growing interest from both academic and industrial sectors due to the increasing
resistance of pathogenic microorganisms to the diverse conventional chemotherapeutics.The
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
40
present study demonstrated that silver nanoparticles synthesized by chemical reduction
method and stabilized with biocompatible polymer chitosan coated on the syringes showed
distinct anti biofilm effect against Staphylococcus aureus can be used in to prevent or to
minimize bacterial infections and will lead to new generation of development of
antimicrobial agents to prevent pathogens infection
REFERENCES
[1].P.T.Ansta, Warner, J. Green Chemistry- Theory and Practice,.
NewYork oxford
University Press, Inc,1996.
[2]. B.J.Baker, J.F.Banfield. Microbial communities in acid mine drainage .FEMS Microbiol
Ecol 44: 139–152 (2003)
[3]. G.K, Druschel , B.J. Baker .,T.M. Gihring, J.F. Banfield
Acid mine drainage.
biogeochemistry at Iron Mountain, California. Geochem Trans.,5, 13–32 (2002)
IJOART
[4].R.Joseph.Prosthetic joint infections: Bane of orthopaedists. Clin Infect Dis., 36, 11571161(2003)
[5].M Parsek and Greenberg.
Sociomicrobiology: the connections between quorum
sensing and biofilms, Trends in Microbiology., 13,3-27, 2004.
[6].R.O,Darouiche, II.. Raad , .S.O
Heard , J.I.Thornby, O.C.Wenker,
A. Gabrielli.
A
comparison of two antimicrobial-impregnated central venous catheters. N.Engl.J.Med.,
340,1–8,1999
[7].G.D.Kamal,M.A Pfaller ., L.E.Rempe,.J.R Jebson. Reduced
intravascular
catheter
infection by antibiotic bonding.A prospective, randomized controlled trial. JAMA., 265,2364–
2368,1999.
[8].RH,Flowers,.K.J.Schwenzer , R.F.Kopel , M.J, Fisch, S.I.Tucker , B.M Farr . Efficacy of
an attachable subcutaneous cuff for the prevention of intravascular catheter-related
infection. JAMA. 261,878–83, 1989
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
41
[9].D.G Maki, .L.A Mermel. .Infections due to infusion therapy. In: Bennett .J.V, Brachman
.P.S(eds). Hospital infections. 4th ed. Philadelphia: Lippincott-Raven; 689-724,1998
[10]. R.N. Jones . Can antimicrobial activity be sustained? An appraisal of orally administered drugs
used for respiratory tract infections. Diagn Microbiol Infect Dis., 27:21–28,1997
[11]. L,K.G Shi, E.T. Neoh, W.Kang.Biomaterials.,27,2440–2449,2008
[12]. P.B Bookstaver, J.C.Williamson ., B.K Tucker ., II,Raad, R.J Sherertz. (2009).Activity
of novel
antibiotic lock solutions in a model against isolates of catheter-related
bloodstream infections. Ann Pharmacother., 43,210-219,2009
[13].M.LW Knetsch, L.H. Koole. New strategies in the development of antimicrobial
coatings: the example of increasing usage of silver and silver nanoparticles. Polymers, 3,340366 (2011)
IJOART
[14] J.Lönn-Stensrud, M.A Landin , T. Benneche, F.C. Petersen ., A..Scheie A. Furanones.
A potential agents for preventing Staphylococcus epidermidis biofilm infections; Antimicrob
Chemother, .63,309-316, 2009
[15]. G. Batoni, G. Maisetta, F.L.Brancatisano ., S.Esin, M.Campa..Use of antimicrobial
peptides
against microbial biofilms: advantages and limits. Curr Med Chem. 18,256-
279,2011
[16].G.
Donelli,
JB.Kaplan.(2007).
I,.Francolini,
D.
Romoli,
E.Guaglianone,
A.Piozzi,
C.Ragunath,
Synergistic activity of dispersin B and cefamandole nafate in
inhibition of staphylococcal
biofilm growth on polyurethanes.
Antimicrob Agents
Chemother., .51,2733–2740,2007
[17].R.Shoshani,M. Ko , M. Chris,G. Yossef. Slow release of nitric oxide from
charged
catheters and its effect on biofilm formation by Escherichia coli. Antimicrob Agents
Chemother., 54,273-279 (2010)
[18].J.L Del Pozo, .M.S. Rouse, J.N.Mandrekar, M.F.Sampedro , J.M. Steckelberg, R.Patel .
Effect of electrical current on the activities of antimicrobial agents against Pseudomonas
aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis biofilms. Antimicrob
Agents Chemother,53,35–40,2009
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
42
[19]. Z. Hazan, J.Zumeris, H. Jacob, H. Raskin, G .Kratysh, M .Vishnia, N .Dror,T. Barliya
T,M.G> Mandel,G. Lavie Effective prevention of microbial biofilm formation on medical
devices by
low-energy surface acoustic waves. Antimicrob Agents Chemother., 50,4144-
4152,2009
[20].S.Perni ,P. Prokopovich,C. Piccirillo,J.Pratten, IP. Parkin, M.Wilson.. Toluidine bluecontaining polymers exhibit potent bactericidal activity when irradiated with red laser light. J
Mater Chem,.19,2715-2723,2009
[21].M,Rai,A.Yadav,A. Gade. .Silver nanoparticles as a new generation of antimicrobials.
Biotechnol Adv, 27,76-83,2009
[22].P.Gong ,H. Li , X. He, K.Wang, J. Hu, S.Zhang, X.Yang . Preparation and antibacterial
activity of Fe3O4 and Ag nanoparticles. Nanotechnology.,18,604-611,2007
[23].S.Tamilvanan, N. Venkateshan, A. Ludwig.The potential of lipid- and polymer-based
drug
IJOART
delivery carriers for eradicating biofilm consortia on device-related nosocomial
infections. J. Control Release.,128,2-22,2008
[24].A. Martinelli, L D'Ilario, I. Francolini, A. Piozzi .Water state effect on drug release from
an
antibiotic loaded polyurethane matrix containing albumin nanoparticles. Int J Pharm.,
407,197-206,2011
[25]. H. Huang, X. Yang, Carbohydr. Res.,339, 2627–2631,2009
[26] H. Huang, Q. Yuan, X. Yang, Colloid Surf. B., 39, 31–37,2004
[27].J.Azeredo,J.W.Sutherland .The use of phages for the removal of infectious biofilms.
Curr
Pharm Biotechnol, 9,261-266,2008
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
43
Table 1 Biofilm inhibition (%) of S.aureus with metallic nanoparticles
Serial No.
Treatment
Biofilm inhibition (%)
1
AgNp
72.8
2
AgNp-CS
80.4
Table .2: Biofilm inhibition (%) of S.aureus with Silver Nanoparticles (AgNps)
Serial No.
Time-Interval (hrs.)
Biofilm inhibition (%)
1
12
9.8
2
24
11.7
36
15.9
48
21.05
60
34.3
72
41.6
3
4
5
IJOART
6
Table 3. Biofilm inhibition (%) of S.aureus with Chitosan coated Silver Nanoparticles (AgNpCS)
Serial No.
Time-Interval (hrs.)
Biofilm inhibition (%)
1
12
11.2
2
24
14.6
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
44
3
36
19.3
4
48
26.1
5
60
40.7
6
72
49.8
Table 4. Effect of Free and Chitosan coated metallic nanoparticles coated syringes against
biofilm matrix biochemical composition of S.aureus.
Total Carbohydrate
Total Protein
Treatment
(mg/mL)
AgNp
16
17
AgNp-CS
14
15
(mg/mL)
IJOART
Figure 3.SEM image of nanoparticles coated syringe
Figure 3 a.Biofilm of Staph.aureus on un coated syringe
Copyright © 2014 SciResPub.
IJOART
International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014
ISSN 2278-7763
45
Figure 3b.SEM image of nanoparticles coated syringe showed disturbed biofilm
IJOART
Copyright © 2014 SciResPub.
IJOART