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Biogenic selenium nanoparticles inhibit Staphylococcus aureus adherence on different surfaces
Accepted Manuscript Title: Biogenic selenium nanoparticles inhibit Staphylococcus aureus adherence on different surfaces Author: Praveen Sonkusre Swaranjit Singh Cameotra PII: DOI: Reference:
S0927-7765(15)30281-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.10.052 COLSUB 7454
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
19-8-2015 30-10-2015 30-10-2015
Please cite this article as: Praveen Sonkusre, Swaranjit Singh Cameotra, Biogenic selenium nanoparticles inhibit Staphylococcus aureus adherence on different surfaces, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.10.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biogenic selenium nanoparticles inhibit Staphylococcus aureus adherence on different surfaces
Praveen Sonkusrea and Swaranjit Singh Cameotraa*
a
Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India
*Corresponding author: Tel.: 0172-6665223; Fax: 0172-2690632/585 E-mail address: [email protected]
1
GRAPHICAL ABSTRACT
2
Abstract The global issue of nosocomial infection is owing to bacterial colonization and biofilm formation on medical devices which primarily affects critically ill and/or immunocompromised patients and also leads to malfunctioning of the devices. Therefore, it is desirable to prevent bacterial colonization on these devices by coating with a non toxic antimicrobial agent or bacterial adherence inhibitor. Here we have shown Bacillus licheniformis JS2 derived selenium nanoparticles (SeNPs) inhibit Staphylococcus aureus adherence and micro-colony formation on polystyrene, glass, and catheter surface. Results indicated that, the coating of these non toxic biogenic SeNPs, at a concentration of 0.5 mg Se/ml, prohibits bacterial load to more than 60 % on glass and catheter surface, when incubated at 4oC for 24 h in phosphate buffered saline. Furthermore, confocal and electron microscopic observations strongly suggested the inhibition of biofilm and micro-colony formation on SeNP coated glass and catheter surfaces when cultured at 37oC for 72 h in a nutrient rich medium. The study suggests that coating of biogenic SeNPs on medical devices could be an alternative approach for prevention of biofilm related infections.
Keywords: Bacillus licheniformis JS2; Staphylococcus aureus; selenium nanoparticles; adherence; micro-colony, biofilm formation
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1.
Introduction A large segment of nosocomial infections are due to bacterial colonization on medical
devices which primarily affects critically ill and/or immuno-compromised patients and also lead to malfunctioning of the devices [1-3]. In these patients, more than 80 % cases of blood stream infection and more than 90 % cases of urinary tract infection originates due to bacterial colonization on vascular and urinary catheter respectively [2, 4]. The surface of these wide range of medical devices, like mechanical heart valves, endotracheal tubes, dialysis tubes, cardiac pacemakers, urinary catheters, central venous catheters, penile prostheses, orthopedic prostheses, contact lenses and more, are not well protected by the host immune system and thus provide nidus for mostly Staphylococcus epidermidis and Staphylococcus aureus infection [3, 5, 6]. Some strains of S. aureus are difficult to treat because of their biofilm forming ability. These biofilms are made up of polymeric exopolysaccharide that acts as a shield and prevent access of drugs to the bacterium and makes it resistant to many commonly used antibiotics [1, 7, 8]. Infection of this Gram-positive cocci can be severe if it occurs on surgical wounds, lungs or in the bloodstream [9]. Synthesis and development of an infectious biofilm on a device involve sequential steps: 1. Association, 2. Adhesion, 3. Micro-colony formation, 4. Biofilm formation and growth [3, 10]. Adhesion and growth of microorganisms generally require exposure of device with body fluids, such as blood, saliva, urine, or other macromolecules [3, 11]. Since the synthesis of biofilm is linked to the adherence of bacteria, application of surface-modifiers or coating of antimicrobial agents like silver and ciprofloxacin showed promising results in reducing the pathogen colonization, biofilm formation and thus deviceassociated infections [3, 12-14]. Selenium compounds like 2,4,6-tri-para-methoxyphenylselenopyrylium chloride, 9para-chlorophenyloctahydroselenoxanthene, and perhydroselenoxanthene are reported to have excellent antibacterial activity against S. aureus [15, 16]. Reports also suggest that selenium nanoparticles are bactericidal, [1, 17] inhibit bacterial growth and biofilm formation and work as a potential material for orthopaedic prostheses [18]. We used biologically synthesized selenium nanoparticles (SeNPs) to study their bacterial adherence inhibiting properties against clinical isolate of Staphylococcus aureus (MTCC 3160) on 3 different surfaces viz., polystyrene, glass surface and human urinary catheter. Intracellular SeNPs were synthesized and extracted from Bacillus licheniformis
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JS2. Earlier we have reported that these NPs are less toxic compare to other form of selenium [19].
2.
Materials and methods
2.1. Microorganism and culture conditions SeNPs were synthesized aerobically by Bacillus licheniformis JS2 strain, isolated from selenium contaminated agricultural soil of the Nawanshahr district (latitude 31°07’ N and longitude 76°08’ E) of Punjab, India. Bacteria were cultured and maintained on tryptic soya broth and agar plates at 37°C [19, 20].
2.2. Chemicals and reagents Tryptic soya agar and broth were purchased from Hi-Media laboratory. Lysozyme, lauryl-sulfate (sodium dodecyl sulfate, SDS), and absolute ethanol were procured from Sigma-Aldrich. Tris-buffer, HCl, 1-octanol, and chloroform were obtained from MerckMillipore. A good quality human urinary catheter (latex) was purchased from the medical store. SYTO 9 green fluorescent nucleic acid stain was purchased from Life Technologies, and Propidium Iodide (PI) was purchased from Calbiochem®, USA. Polystyrene plates were obtained from Thermo Scientific Nunc®, Denmark. Tissue culture treated cell imaging dish was purchased from Eppendorf, Germany. Type II Millipore water was used in all the experiments.
2.3. Synthesis and extraction of selenium nanoparticles Selenium nanoparticles were synthesised aerobically by Bacillus licheniformis JS2 under 1.8 mM sodium selenite stress, and extracted by using our previously reported protocol [19].
2.4. Quantification of selenium SeNPs were digested overnight in nitric acid (3 parts): perchloric acid (1 part) solution. Digested samples were analyzed in a Shimadzu AA-6800 atomic absorption spectrophotometer to quantitate selenium. Selenium cathode lamp with air-acetylene (oxidizing) flame was used to measure selenium at 196 nm.
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2.5. Coating of SeNPs and adhesion of S. aureus on polystyrene surface 200 µl of SeNPs were filled in each well of 96 well flat bottom tissue culture grade polystyrene plates (Nunc - Delta surface) at a concentration of 0.5 mg Se/ml and 0.1 mg Se/ml under sterile conditions. The plate was kept at 4°C for 24 h to allow coating of SeNPs to the surface. Unattached NPs were pipetted out, and wells were rinsed 3 times with distilled water to remove the loosely bound NPs. The plate was dried for a few minutes in a sterile environment. Overnight grown culture of S. aureus was collected and washed 3 times with phosphate buffered saline (PBS) (pH 7.4), by centrifugation at 1500 x g for 10 min. 100 µl of 0.2 OD600 culture was added to each well and kept at 4°C for 24 h. Unattached cells were removed by 3 gentle washes with PBS. Adhered bacteria were fixed with 200 µl of 99 % methanol. After 15 min of fixing, the solution was drained and the plate was dried for 30 min. 200 µl of 1 % crystal violet stain was added and kept for 5 min. Excess stain was removed by rinsing the plate with distilled water and the plate was dried for 30 min. Dye bound to the adhered bacteria was solubilized by adding 200 µl of 33 % glacial acetic acid and read at 590 nm on a BioTek Power Wave Microplate reader.
2.6. Coating of SeNPs and adhesion of S. aureus on glass surface SeNPs at a concentration of 0.5 mg Se/ml were used for coating the glass surface of cell imaging dishes (CIDs) as mentioned above. Overnight grown culture of S. aureus was collected and washed 3 times with PBS by centrifugation at 1500 x g for 10 min. 250 µl of 0.4 OD600 culture was filled in both NP coated and uncoated CIDs and kept at 4°C for 24 h to allow adherence. After 24 h, supernatant of the dish was discarded. Each dish was washed 3 times with PBS to remove loosely bound cells, stained with SYTO 9 and PI and observed under confocal microscope. In another set, 200 µl of PBS was added to CIDs and sonicated in a sonicator water bath for 2-3 min. Sonicated sample was collected in a microcentrifuge tube for each plate separately. This step was repeated twice with 200 µl and 100 µl PBS respectively. Different dilutions of the collected samples were prepared, and 50 µl from each was spread on TSA plates for CFU count. The experiments were repeated in triplicates.
2.7. Coating of SeNPs and adhesion of S. aureus on urinary catheter Sections of human urinary catheter were coated with 0.5 mg Se/ml SeNPs as mentioned above. NP coated and uncoated catheter sections of equal dimensions were dipped in 1 ml PBS containing 0.4 OD600 S. aureus culture for 24 h at 4°C in a 12 well microtiter plate to 6
allow adherence. After 24 h, supernatant from the wells were discarded. Each section was washed 3 times with PBS to remove loosely bound cells and processed for scanning electron microscopy. In another set, 500 µl PBS was added to each well and sonicated in a sonicator water bath for 3-4 min. The resulting supernatant was collected in a microcentrifuge tube and the step was performed again. Different dilutions of the collected samples were prepared and 50 µl from each was spread on TSA plates for CFU count. Experiments were conducted in triplicates.
2.8. Biofilm formation by S. aureus on glass and catheter surface in tryptic soya broth (TSB) 2 ml of 0.6 OD600 culture in TSB medium was loaded separately on SeNP coated and uncoated CIDs and catheter sections. CIDs and 12 well plates containing the catheter sections were kept static at 37°C for 3 days. 50 % of the medium was replaced after regular interval of 24 h. After 72 h CIDs were gently washed with PBS, stained with fluorescent dyes and observed under confocal microscope. Catheter sections were washed 3 times with PBS and processed for scanning electron microscopy using the standard fixation procedure for bacterial visualization. Images of nanoparticles were obtained on a Zeiss EVO 40 SEM microscope. Another set of catheter sections was used for the CFU count as mentioned above.
2.9. Confocal microscopy After successive washes with PBS, each CID was layered with 70 µl of PBS. 10 µl of 0.1 M SYTO 9 stain was added in dark and kept for 5 min. Samples were rinsed with PBS, 10 µl of Propidium Iodide (30 µg/ml) was added and incubated in the dark for another 5 min. Rinsed with 3 rounds of PBS and observed under Nikon A1R confocal microscope in 100 X oil immersion with 488 and 514 nm argon laser. Z-scans were taken with an increment of 0.1 µm for three-dimensional image projection of biofilm.
2.10. Statistical analysis The data represented here are the mean ± SD from at least 3 independent experiments. Statistical significances were analyzed using two-tailed Student’s t-test.
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3.
Results Bacillus licheniformis JS2 strain was used for intracellular conversion of toxic Selenite
ions (Se+4) into nontoxic elemental SeNPs (Se0), aerobically. SeNPs were extracted and purified by complete bacterial cell lysis using lysozyme and French press followed by consecutive wash with Tris-Cl and extraction with water-octanol 2 phase system.
3.1. Adherence of S. aureus on SeNPs coated polystyrene surface Bacterial adherence inhibiting property of B. licheniformis derived selenium nanoparticles was performed against a pathogenic strain of S. aureus (MTCC 3160, isolated from human carbuncle) on tissue culture grade flat bottom 96 well plates, meant for adherent cell growth. Wells were first coated with two different concentrations of SeNPs, viz., 0.1 and 0.5 mg Se/ml (Fig 1a) and then the bacterial sample was loaded. Number of adhered bacteria was measured indirectly by following standard crystal violet staining for bacterial biofilm detection. An inverse relationship was observed between the adherence of bacteria and the concentration of SeNPs. A significant difference with *p value < 0.05 was observed between the relative cell number adhered on SeNPs coated (0.5 mg Se/ml) and control wells. Whereas, no significant difference was observed on 0.1 mg Se/ml SeNP coated wells (Fig. 1b). Experiments were performed in 3 different replicates. And the concentration of 0.5 mg Se/ml SeNPs was selected for further studies.
3.2. Adhesion of S. aureus on SeNP coated glass surface Tissue culture grade live cell imaging dish (CID) was used as a glass surface to study adherence of S. aureus. Bacteria were allowed to adhere on SeNPs coated and uncoated CIDs in PBS at 4oC and were visualized by confocal microscope. Staining with SYTO 9 (green fluorescent nucleic acid stain) and PI (dead cell specific red-fluorescent nuclear and chromosome counter-stain) showed absence of dead cells. Number of adhered bacteria on normal CID was more and densely populated compared to SeNP coated CID (Fig. 2a & 2b). We have shown only the green florescent images taken with 488 nm argon laser (PI stained data is not shown). The results were revalidated with the CFU count by plating the different dilutions of adhered bacterial cells on tryptic soya agar plates; clearly indicating more than 65 % reduction in bacterial cell adherence (Fig. 2c).
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3.3. S. aureus colonization and adherence on SeNP coated glass surface in nutrient rich tryptic soya broth medium Bacteria were allowed to grow at physiological temperature on SeNP coated and uncoated CIDs in 2 ml TSB medium for 3 days. After successive washes with PBS and staining with SYTO 9 and PI, confocal microscopy was performed to see the biofilm forming ability of the bacterium on the glass surface in a nutrient rich medium. Results showed a thick layer of bacteria in normal glass surface with very few dead cells, as evident by PI staining, suggesting the formation of biofilm (Fig. 3a). On the other hand, less number of bacteria were visible on the SeNP coated glass surface (Fig. 3b). Results suggest that the SeNPs are able to obstruct S. aureus adherence on glass surface significantly. 3.4. Adhesion and biofilm formation of S. aureus on SeNP coated catheter surface SeNP coated and uncoated catheter fragments of equal dimensions were dipped in PBS containing 0.4 OD600 S. aureus culture for 24 h at 4oC to allow adherence. The inner wall of the catheter was visualized at 5000 X magnification in Scanning Electron Microscope. Images clearly showed that the numbers of adhered bacteria were more on the normal catheter surface compared to SeNP coated surface (Fig. 4a & 4b). To support the above results CFU count of adhered bacteria were also performed on TSA plates; showing the significant difference between the two cases (Fig. 4c). Biofilm forming ability of S. aureus was tested in TSB medium. Bacteria were cultured in static condition along with equal dimension catheter sections at 37oC for 72 h in TSB medium. Large numbers of aggregated bacteria were observed in SEM images of uncoated catheter surface. Whereas, less and isolated bacteria were observed on the nanoparticle coated catheter. To support the above results, CFU counts of adhered bacteria were also performed on TSA plates. A significantly less number of bacteria were observed on the NP coated surface compared to the uncoated surface, *p < 0.05.
4.
Discussion Nosocomial infection is a global issue that mainly affects immunocompromised
patients and medical devices. Microbial colonization and biofilm formation provide resistance to bacteria by obstructing drug bacterium interaction and thus become difficult to eradicate [21-23]. As a result of which patient undergoes frequent replacement of different prostheses. Efforts have been taken to resolve this issue, but not much success has been achieved. In such cases, coating of antimicrobial agents on prostheses is generally preferred 9
to prevent the infection. Several types of coatings were tried like synthetic polymers, metals and alloys, carbon based materials, and biological materials [24-27]. Among these, coating of silver with antibiotics is preferred on medical devices to combat against S. aureus and other nosocomial infections [3]. Recently, metalloid and metal based NPs are also studied extensively for antimicrobial properties. Materials in the form of NPs exhibit completely different physical and chemical properties when compared to the corresponding bulk material. The high surface to volume ratio allows more active sites for interaction with biological entities. So far silver nanoparticles are reported to have the best antimicrobial properties in metal segment against pathogenic bacteria [5, 28]. Apart from silver, selenium is also reported to have excellent antimicrobial property. Chemically synthesized SeNPs are reported to inhibit bacterial growth and biofilm formation and prevent bacterial colonization on orthopedic prostheses [1, 17, 18, 29]. The reason behind antibacterial activity could be oxidative stress [30]. Chudobova et. al. also showed that the antibacterial activity of NPs is size dependent, smaller sized SeNP (50-100 nm) have more antibacterial activity compare to bigger sized silver NP (200-300 nm) [29]. Most of the earlier studies were focused on the bactericidal activity of Se and not specifically on bacterial adherence. Since the nosocomial infection is related to bacterial colonization and biofilm formation on medical devices, our work has focused on bacterial adherence and biofilm inhibition properties of biogenic SeNPs. In an earlier report from our lab, Bacillus licheniformis JS2 derived intracellular biogenic SeNPs were extracted, purified, and characterized for their shape, size, and purity with Dynamic Light Scattering, Scanning and Transmission Electron Microscopy, Fourier Transform Infrared Spectroscopy, Energy Dispersive X-ray Spectroscopy, and SDSPAGE/Silver staining. Moreover, these SeNPs were stable under physiological buffered condition and were non-toxic to the non-cancerous human peripheral blood mononuclear cells [19]. Here, we have demonstrated the bacterial adherence inhibiting properties of SeNPs against a most predominant hospital contaminant, S. aureus, on 3 different surfaces. Coating of these NP reduces the bacterial load significantly to more than 60 % on glass and catheter surface, both in PBS and in the nutrient rich TSB medium. It is interesting to note that S. aureus is not able to form micro-colonies on catheter surface in the presence of SeNPs showing the inhibition of the critical steps of bacterial colonization and biofilm formation by SeNPs. Similar results were obtained on the glass surface in confocal microscopy where 10
thick and dense biofilm was observed on the uncoated cell imaging dish and a thin, and sparsely populated population was observed on SeNP coated CID. These results clearly indicate the S. aureus biofilm inhibiting activity of biogenic SeNPs. The green synthesis approach addressed here for selenium nanoparticles production is simple, eco-friendly, cost effective, and the resultant nanoparticles are non toxic and highly stable [19]. However, the limitation with this system is to control NP size and surface properties. Probably because of this we observed uneven coating of NPs on catheter surface in scanning electron microscopy. This could have lead to bacterial entrapment into the bunch of NPs and thus showing more bacterial count. We are expecting lesser bacterial count on smooth and uniform size coating of SeNPs. Antibacterial and anti-adhesion activity against other pathogens is further needs to be investigated.
5.
Conclusion As per our knowledge, this is the first report to show a comparison in the bacterial
adherence inhibition by biogenic selenium nanoparticles on 3 different surfaces. The results clearly demonstrate that B. licheniformis derived non toxic biogenic SeNPs are promising bacterial adherence inhibiting agent against the employed Gram positive Staphylococcus aureus. The activity of SeNPs can be increased when subjected in combination with other antimicrobial agents.
Acknowledgement We are thankful to the Director, Institute of Microbial Technology, a constituent laboratory of Council of Scientific and Industrial Research (CSIR) for providing excellent infrastructure and facility. This work was financially supported by the Department of Biotechnology, Government of India & CSIR. P.S. was provided research fellowship by CSIR.
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References [1] P.A. Tran, T.J. Webster, Selenium nanoparticles inhibit Staphylococcus aureus growth, Int J Nanomedicine, 6 (2011) 1553-1558. [2] R.O. Darouiche, Device-associated infections: a macroproblem that starts with microadherence, Clin Infect Dis, 33 (2001) 1567-1572. [3] G. Reid, Biofilms in infectious disease and on medical devices, Int J Antimicrob Agents, 11 (1999) 223-226; discussion 237-229. [4] M.J. Richards, J.R. Edwards, D.H. Culver, R.P. Gaynes, Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System, Crit Care Med, 27 (1999) 887-892. [5] R.J. McLean, A.A. Hussain, M. Sayer, P.J. Vincent, D.J. Hughes, T.J. Smith, Antibacterial activity of multilayer silver-copper surface films on catheter material, Can J Microbiol, 39 (1993) 895-899. [6] R.R. Muder, C. Brennen, J.D. Rihs, M.M. Wagener, A. Obman, J.E. Stout, V.L. Yu, Isolation of Staphylococcus aureus from the urinary tract: association of isolation with symptomatic urinary tract infection and subsequent staphylococcal bacteremia, Clin Infect Dis, 42 (2006) 46-50. [7] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections, Science, 284 (1999) 1318-1322. [8] P.S. Stewart, J.W. Costerton, Antibiotic resistance of bacteria in biofilms, Lancet, 358 (2001) 135-138. [9] L.R. Martinez, G. Han, M. Chacko, M.R. Mihu, M. Jacobson, P. Gialanella, A.J. Friedman, J.D. Nosanchuk, J.M. Friedman, Antimicrobial and healing efficacy of sustained release nitric oxide nanoparticles against Staphylococcus aureus skin infection, J Invest Dermatol, 129 (2009) 2463-2469. [10] J.W. Costerton, K.J. Cheng, G.G. Geesey, T.I. Ladd, J.C. Nickel, M. Dasgupta, T.J. Marrie, Bacterial biofilms in nature and disease, Annu Rev Microbiol, 41 (1987) 435-464. [11] A.G. Gristina, Biomaterial-centered infection: microbial adhesion versus tissue integration, Science, 237 (1987) 1588-1595. [12] H. Liedberg, P. Ekman, T. Lundeberg, Pseudomonas aeruginosa: adherence to and growth on different urinary catheter coatings, Int Urol Nephrol, 22 (1990) 487-492. [13] J.R. Johnson, P.L. Roberts, R.J. Olsen, K.A. Moyer, W.E. Stamm, Prevention of catheter-associated urinary tract infection with a silver oxide-coated urinary catheter: clinical and microbiologic correlates, J Infect Dis, 162 (1990) 1145-1150. 12
[14] A.J. Schaeffer, K.O. Story, S.M. Johnson, Effect of silver oxide/trichloroisocyanuric acid antimicrobial urinary drainage system on catheter-associated bacteriuria, J Urol, 139 (1988) 69-73. [15] H. Kucukbay, R. Durmaz, E. Orhan, S. Gunal, Synthesis, antibacterial and antifungal activities of electron-rich olefins derived benzimidazole compounds, Farmaco, 58 (2003) 431-437. [16] M. Pietka-Ottlik, H. Wojtowicz-Mlochowska, K. Kolodziejczyk, E. Piasecki, J. Mlochowski, New organoselenium compounds active against pathogenic bacteria, fungi and viruses, Chem Pharm Bull (Tokyo), 56 (2008) 1423-1427. [17] Q. Wang, T.J. Webster, Short communication: inhibiting biofilm formation on paper towels through the use of selenium nanoparticles coatings, Int J Nanomedicine, 8 (2013) 407411. [18] Q. Wang, T.J. Webster, Nanostructured selenium for preventing biofilm formation on polycarbonate medical devices, J Biomed Mater Res A, 100 (2012) 3205-3210. [19] P. Sonkusre, R. Nanduri, P. Gupta, S.S. Cameotra, Improved extraction of intracellular biogenic selenium nanoparticles and their specificity for cancer chemoprevention, J Nanomed Nanotechnol, 5 (2014) 194-202. [20] S. Dhanjal, S.S. Cameotra, Selenite stress elicits physiological adaptations in Bacillus sp. (strain JS-2), J Microbiol Biotechnol, 21 (2011) 1184-1192. [21] P. Gilbert, D.G. Allison, A.J. McBain, Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?, Symp Ser Soc Appl Microbiol, (2002) 98S-110S. [22] T.F. Mah, G.A. O'Toole, Mechanisms of biofilm resistance to antimicrobial agents, Trends Microbiol, 9 (2001) 34-39. [23] R. Singh, P. Ray, A. Das, M. Sharma, Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study, J Med Microbiol, 58 (2009) 1067-1073. [24] G. Kotzar, M. Freas, P. Abel, A. Fleischman, S. Roy, C. Zorman, J.M. Moran, J. Melzak, Evaluation of MEMS materials of construction for implantable medical devices, Biomaterials, 23 (2002) 2737-2750. [25] X. Ding, C. Yang, T.P. Lim, L.Y. Hsu, A.C. Engler, J.L. Hedrick, Y.Y. Yang, Antibacterial and antifouling catheter coatings using surface grafted PEG-b-cationic polycarbonate diblock copolymers, Biomaterials, 33 (2012) 6593-6603. [26] D. Graiver, R.L. Durall, T. Okada, Surface morphology and friction coefficient of various types of Foley catheter, Biomaterials, 14 (1993) 465-469. 13
[27] D. Fraenkel, C. Rickard, P. Thomas, J. Faoagali, N. George, R. Ware, A prospective, randomized trial of rifampicin-minocycline-coated and silver-platinum-carbon-impregnated central venous catheters, Crit Care Med, 34 (2006) 668-675. [28] D.R. Monteiro, L.F. Gorup, A.S. Takamiya, A.C. Ruvollo-Filho, E.R. de Camargo, D.B. Barbosa, The growing importance of materials that prevent microbial adhesion: antimicrobial effect of medical devices containing silver, Int J Antimicrob Agents, 34 (2009) 103-110. [29] D. Chudobova, K. Cihalova, S. Dostalova, B. Ruttkay-Nedecky, M.A. Rodrigo, K. Tmejova, P. Kopel, L. Nejdl, J. Kudr, J. Gumulec, S. Krizkova, J. Kynicky, R. Kizek, V. Adam, Comparison of the effects of silver phosphate and selenium nanoparticles on Staphylococcus aureus growth reveals potential for selenium particles to prevent infection, FEMS Microbiol Lett, 351 (2014) 195-201. [30] L. Yan, Z. Gu, Y. Zhao, Chemical mechanisms of the toxicological properties of nanomaterials: generation of intracellular reactive oxygen species, Chem Asian J, 8 (2013) 2342-2353.
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Figure legends
Figure 1. (a) SeNP coated 96 well flat bottom polystyrene plate. (b) Absorbance of crystal violet stain at 590 nm corresponds to the number of bacteria present. Significant reduction (*p < 0.05) in bacterial adherence was observed between the control and SeNP (0.5 mg Se/ml) coated surface.
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Figure 2. Confocal microscopy was performed at 100 X oil immersion with 488 nm argon laser. Staining of bacteria with SYTO 9- nucleic acid stain, showing the (a) accumulation of less bacterial population on the SeNP coated glass surface (b) and dense population on uncoated surface. (c) CFU count showing the significant difference in adhered bacterial cell population of the two sets with *p < 0.05. Experiments were performed in triplicates.
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Figure 3. Three dimensional view of biofilm formed by S. aureus on (a) SeNP coated (b) and uncoated glass surface after 3 days of growth in TSB medium. Cells were stained with SYTO 9 and Propidium Iodide for viable and dead cell discrimination. Confocal microscopy was performed at 100 X oil immersion with 488 and 514 nm lasers. 17
Figure 4. SEM images of SeNP coated and uncoated catheter sections incubated with 0.4 OD600 S. aureus culture at 4oC for 24 h. (a) Images showing very few adhered bacteria, marked in red (rest is the SeNPs), on SeNP coated catheter compare to (b) uncoated catheter. (c) CFU count showing the significant difference in adhered bacterial cell population in these two experiment sets with *p < 0.05. Experiments were performed in triplicates.
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Figure 5. SEM images of SeNP coated and uncoated catheter sections incubated at 37oC with 0.6 OD600 S. aureus culture in TSB medium for 72 h. (a) Images showing few isolated, adhered bacteria, indicated by arrows, on SeNP coated catheter whereas, (b) more bacteria with microcolonies were observed on uncoated catheter. These microcolonies will act as precursor for biofilm formation. (c) CFU count confirms the SEM results showing significant difference in adhered cell population between the two sets with *p < 0.05. Experiments were performed in triplicates.
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S0927-7765(15)30281-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.10.052 COLSUB 7454
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
19-8-2015 30-10-2015 30-10-2015
Please cite this article as: Praveen Sonkusre, Swaranjit Singh Cameotra, Biogenic selenium nanoparticles inhibit Staphylococcus aureus adherence on different surfaces, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.10.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biogenic selenium nanoparticles inhibit Staphylococcus aureus adherence on different surfaces
Praveen Sonkusrea and Swaranjit Singh Cameotraa*
a
Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India
*Corresponding author: Tel.: 0172-6665223; Fax: 0172-2690632/585 E-mail address: [email protected]
1
GRAPHICAL ABSTRACT
2
Abstract The global issue of nosocomial infection is owing to bacterial colonization and biofilm formation on medical devices which primarily affects critically ill and/or immunocompromised patients and also leads to malfunctioning of the devices. Therefore, it is desirable to prevent bacterial colonization on these devices by coating with a non toxic antimicrobial agent or bacterial adherence inhibitor. Here we have shown Bacillus licheniformis JS2 derived selenium nanoparticles (SeNPs) inhibit Staphylococcus aureus adherence and micro-colony formation on polystyrene, glass, and catheter surface. Results indicated that, the coating of these non toxic biogenic SeNPs, at a concentration of 0.5 mg Se/ml, prohibits bacterial load to more than 60 % on glass and catheter surface, when incubated at 4oC for 24 h in phosphate buffered saline. Furthermore, confocal and electron microscopic observations strongly suggested the inhibition of biofilm and micro-colony formation on SeNP coated glass and catheter surfaces when cultured at 37oC for 72 h in a nutrient rich medium. The study suggests that coating of biogenic SeNPs on medical devices could be an alternative approach for prevention of biofilm related infections.
Keywords: Bacillus licheniformis JS2; Staphylococcus aureus; selenium nanoparticles; adherence; micro-colony, biofilm formation
3
1.
Introduction A large segment of nosocomial infections are due to bacterial colonization on medical
devices which primarily affects critically ill and/or immuno-compromised patients and also lead to malfunctioning of the devices [1-3]. In these patients, more than 80 % cases of blood stream infection and more than 90 % cases of urinary tract infection originates due to bacterial colonization on vascular and urinary catheter respectively [2, 4]. The surface of these wide range of medical devices, like mechanical heart valves, endotracheal tubes, dialysis tubes, cardiac pacemakers, urinary catheters, central venous catheters, penile prostheses, orthopedic prostheses, contact lenses and more, are not well protected by the host immune system and thus provide nidus for mostly Staphylococcus epidermidis and Staphylococcus aureus infection [3, 5, 6]. Some strains of S. aureus are difficult to treat because of their biofilm forming ability. These biofilms are made up of polymeric exopolysaccharide that acts as a shield and prevent access of drugs to the bacterium and makes it resistant to many commonly used antibiotics [1, 7, 8]. Infection of this Gram-positive cocci can be severe if it occurs on surgical wounds, lungs or in the bloodstream [9]. Synthesis and development of an infectious biofilm on a device involve sequential steps: 1. Association, 2. Adhesion, 3. Micro-colony formation, 4. Biofilm formation and growth [3, 10]. Adhesion and growth of microorganisms generally require exposure of device with body fluids, such as blood, saliva, urine, or other macromolecules [3, 11]. Since the synthesis of biofilm is linked to the adherence of bacteria, application of surface-modifiers or coating of antimicrobial agents like silver and ciprofloxacin showed promising results in reducing the pathogen colonization, biofilm formation and thus deviceassociated infections [3, 12-14]. Selenium compounds like 2,4,6-tri-para-methoxyphenylselenopyrylium chloride, 9para-chlorophenyloctahydroselenoxanthene, and perhydroselenoxanthene are reported to have excellent antibacterial activity against S. aureus [15, 16]. Reports also suggest that selenium nanoparticles are bactericidal, [1, 17] inhibit bacterial growth and biofilm formation and work as a potential material for orthopaedic prostheses [18]. We used biologically synthesized selenium nanoparticles (SeNPs) to study their bacterial adherence inhibiting properties against clinical isolate of Staphylococcus aureus (MTCC 3160) on 3 different surfaces viz., polystyrene, glass surface and human urinary catheter. Intracellular SeNPs were synthesized and extracted from Bacillus licheniformis
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JS2. Earlier we have reported that these NPs are less toxic compare to other form of selenium [19].
2.
Materials and methods
2.1. Microorganism and culture conditions SeNPs were synthesized aerobically by Bacillus licheniformis JS2 strain, isolated from selenium contaminated agricultural soil of the Nawanshahr district (latitude 31°07’ N and longitude 76°08’ E) of Punjab, India. Bacteria were cultured and maintained on tryptic soya broth and agar plates at 37°C [19, 20].
2.2. Chemicals and reagents Tryptic soya agar and broth were purchased from Hi-Media laboratory. Lysozyme, lauryl-sulfate (sodium dodecyl sulfate, SDS), and absolute ethanol were procured from Sigma-Aldrich. Tris-buffer, HCl, 1-octanol, and chloroform were obtained from MerckMillipore. A good quality human urinary catheter (latex) was purchased from the medical store. SYTO 9 green fluorescent nucleic acid stain was purchased from Life Technologies, and Propidium Iodide (PI) was purchased from Calbiochem®, USA. Polystyrene plates were obtained from Thermo Scientific Nunc®, Denmark. Tissue culture treated cell imaging dish was purchased from Eppendorf, Germany. Type II Millipore water was used in all the experiments.
2.3. Synthesis and extraction of selenium nanoparticles Selenium nanoparticles were synthesised aerobically by Bacillus licheniformis JS2 under 1.8 mM sodium selenite stress, and extracted by using our previously reported protocol [19].
2.4. Quantification of selenium SeNPs were digested overnight in nitric acid (3 parts): perchloric acid (1 part) solution. Digested samples were analyzed in a Shimadzu AA-6800 atomic absorption spectrophotometer to quantitate selenium. Selenium cathode lamp with air-acetylene (oxidizing) flame was used to measure selenium at 196 nm.
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2.5. Coating of SeNPs and adhesion of S. aureus on polystyrene surface 200 µl of SeNPs were filled in each well of 96 well flat bottom tissue culture grade polystyrene plates (Nunc - Delta surface) at a concentration of 0.5 mg Se/ml and 0.1 mg Se/ml under sterile conditions. The plate was kept at 4°C for 24 h to allow coating of SeNPs to the surface. Unattached NPs were pipetted out, and wells were rinsed 3 times with distilled water to remove the loosely bound NPs. The plate was dried for a few minutes in a sterile environment. Overnight grown culture of S. aureus was collected and washed 3 times with phosphate buffered saline (PBS) (pH 7.4), by centrifugation at 1500 x g for 10 min. 100 µl of 0.2 OD600 culture was added to each well and kept at 4°C for 24 h. Unattached cells were removed by 3 gentle washes with PBS. Adhered bacteria were fixed with 200 µl of 99 % methanol. After 15 min of fixing, the solution was drained and the plate was dried for 30 min. 200 µl of 1 % crystal violet stain was added and kept for 5 min. Excess stain was removed by rinsing the plate with distilled water and the plate was dried for 30 min. Dye bound to the adhered bacteria was solubilized by adding 200 µl of 33 % glacial acetic acid and read at 590 nm on a BioTek Power Wave Microplate reader.
2.6. Coating of SeNPs and adhesion of S. aureus on glass surface SeNPs at a concentration of 0.5 mg Se/ml were used for coating the glass surface of cell imaging dishes (CIDs) as mentioned above. Overnight grown culture of S. aureus was collected and washed 3 times with PBS by centrifugation at 1500 x g for 10 min. 250 µl of 0.4 OD600 culture was filled in both NP coated and uncoated CIDs and kept at 4°C for 24 h to allow adherence. After 24 h, supernatant of the dish was discarded. Each dish was washed 3 times with PBS to remove loosely bound cells, stained with SYTO 9 and PI and observed under confocal microscope. In another set, 200 µl of PBS was added to CIDs and sonicated in a sonicator water bath for 2-3 min. Sonicated sample was collected in a microcentrifuge tube for each plate separately. This step was repeated twice with 200 µl and 100 µl PBS respectively. Different dilutions of the collected samples were prepared, and 50 µl from each was spread on TSA plates for CFU count. The experiments were repeated in triplicates.
2.7. Coating of SeNPs and adhesion of S. aureus on urinary catheter Sections of human urinary catheter were coated with 0.5 mg Se/ml SeNPs as mentioned above. NP coated and uncoated catheter sections of equal dimensions were dipped in 1 ml PBS containing 0.4 OD600 S. aureus culture for 24 h at 4°C in a 12 well microtiter plate to 6
allow adherence. After 24 h, supernatant from the wells were discarded. Each section was washed 3 times with PBS to remove loosely bound cells and processed for scanning electron microscopy. In another set, 500 µl PBS was added to each well and sonicated in a sonicator water bath for 3-4 min. The resulting supernatant was collected in a microcentrifuge tube and the step was performed again. Different dilutions of the collected samples were prepared and 50 µl from each was spread on TSA plates for CFU count. Experiments were conducted in triplicates.
2.8. Biofilm formation by S. aureus on glass and catheter surface in tryptic soya broth (TSB) 2 ml of 0.6 OD600 culture in TSB medium was loaded separately on SeNP coated and uncoated CIDs and catheter sections. CIDs and 12 well plates containing the catheter sections were kept static at 37°C for 3 days. 50 % of the medium was replaced after regular interval of 24 h. After 72 h CIDs were gently washed with PBS, stained with fluorescent dyes and observed under confocal microscope. Catheter sections were washed 3 times with PBS and processed for scanning electron microscopy using the standard fixation procedure for bacterial visualization. Images of nanoparticles were obtained on a Zeiss EVO 40 SEM microscope. Another set of catheter sections was used for the CFU count as mentioned above.
2.9. Confocal microscopy After successive washes with PBS, each CID was layered with 70 µl of PBS. 10 µl of 0.1 M SYTO 9 stain was added in dark and kept for 5 min. Samples were rinsed with PBS, 10 µl of Propidium Iodide (30 µg/ml) was added and incubated in the dark for another 5 min. Rinsed with 3 rounds of PBS and observed under Nikon A1R confocal microscope in 100 X oil immersion with 488 and 514 nm argon laser. Z-scans were taken with an increment of 0.1 µm for three-dimensional image projection of biofilm.
2.10. Statistical analysis The data represented here are the mean ± SD from at least 3 independent experiments. Statistical significances were analyzed using two-tailed Student’s t-test.
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3.
Results Bacillus licheniformis JS2 strain was used for intracellular conversion of toxic Selenite
ions (Se+4) into nontoxic elemental SeNPs (Se0), aerobically. SeNPs were extracted and purified by complete bacterial cell lysis using lysozyme and French press followed by consecutive wash with Tris-Cl and extraction with water-octanol 2 phase system.
3.1. Adherence of S. aureus on SeNPs coated polystyrene surface Bacterial adherence inhibiting property of B. licheniformis derived selenium nanoparticles was performed against a pathogenic strain of S. aureus (MTCC 3160, isolated from human carbuncle) on tissue culture grade flat bottom 96 well plates, meant for adherent cell growth. Wells were first coated with two different concentrations of SeNPs, viz., 0.1 and 0.5 mg Se/ml (Fig 1a) and then the bacterial sample was loaded. Number of adhered bacteria was measured indirectly by following standard crystal violet staining for bacterial biofilm detection. An inverse relationship was observed between the adherence of bacteria and the concentration of SeNPs. A significant difference with *p value < 0.05 was observed between the relative cell number adhered on SeNPs coated (0.5 mg Se/ml) and control wells. Whereas, no significant difference was observed on 0.1 mg Se/ml SeNP coated wells (Fig. 1b). Experiments were performed in 3 different replicates. And the concentration of 0.5 mg Se/ml SeNPs was selected for further studies.
3.2. Adhesion of S. aureus on SeNP coated glass surface Tissue culture grade live cell imaging dish (CID) was used as a glass surface to study adherence of S. aureus. Bacteria were allowed to adhere on SeNPs coated and uncoated CIDs in PBS at 4oC and were visualized by confocal microscope. Staining with SYTO 9 (green fluorescent nucleic acid stain) and PI (dead cell specific red-fluorescent nuclear and chromosome counter-stain) showed absence of dead cells. Number of adhered bacteria on normal CID was more and densely populated compared to SeNP coated CID (Fig. 2a & 2b). We have shown only the green florescent images taken with 488 nm argon laser (PI stained data is not shown). The results were revalidated with the CFU count by plating the different dilutions of adhered bacterial cells on tryptic soya agar plates; clearly indicating more than 65 % reduction in bacterial cell adherence (Fig. 2c).
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3.3. S. aureus colonization and adherence on SeNP coated glass surface in nutrient rich tryptic soya broth medium Bacteria were allowed to grow at physiological temperature on SeNP coated and uncoated CIDs in 2 ml TSB medium for 3 days. After successive washes with PBS and staining with SYTO 9 and PI, confocal microscopy was performed to see the biofilm forming ability of the bacterium on the glass surface in a nutrient rich medium. Results showed a thick layer of bacteria in normal glass surface with very few dead cells, as evident by PI staining, suggesting the formation of biofilm (Fig. 3a). On the other hand, less number of bacteria were visible on the SeNP coated glass surface (Fig. 3b). Results suggest that the SeNPs are able to obstruct S. aureus adherence on glass surface significantly. 3.4. Adhesion and biofilm formation of S. aureus on SeNP coated catheter surface SeNP coated and uncoated catheter fragments of equal dimensions were dipped in PBS containing 0.4 OD600 S. aureus culture for 24 h at 4oC to allow adherence. The inner wall of the catheter was visualized at 5000 X magnification in Scanning Electron Microscope. Images clearly showed that the numbers of adhered bacteria were more on the normal catheter surface compared to SeNP coated surface (Fig. 4a & 4b). To support the above results CFU count of adhered bacteria were also performed on TSA plates; showing the significant difference between the two cases (Fig. 4c). Biofilm forming ability of S. aureus was tested in TSB medium. Bacteria were cultured in static condition along with equal dimension catheter sections at 37oC for 72 h in TSB medium. Large numbers of aggregated bacteria were observed in SEM images of uncoated catheter surface. Whereas, less and isolated bacteria were observed on the nanoparticle coated catheter. To support the above results, CFU counts of adhered bacteria were also performed on TSA plates. A significantly less number of bacteria were observed on the NP coated surface compared to the uncoated surface, *p < 0.05.
4.
Discussion Nosocomial infection is a global issue that mainly affects immunocompromised
patients and medical devices. Microbial colonization and biofilm formation provide resistance to bacteria by obstructing drug bacterium interaction and thus become difficult to eradicate [21-23]. As a result of which patient undergoes frequent replacement of different prostheses. Efforts have been taken to resolve this issue, but not much success has been achieved. In such cases, coating of antimicrobial agents on prostheses is generally preferred 9
to prevent the infection. Several types of coatings were tried like synthetic polymers, metals and alloys, carbon based materials, and biological materials [24-27]. Among these, coating of silver with antibiotics is preferred on medical devices to combat against S. aureus and other nosocomial infections [3]. Recently, metalloid and metal based NPs are also studied extensively for antimicrobial properties. Materials in the form of NPs exhibit completely different physical and chemical properties when compared to the corresponding bulk material. The high surface to volume ratio allows more active sites for interaction with biological entities. So far silver nanoparticles are reported to have the best antimicrobial properties in metal segment against pathogenic bacteria [5, 28]. Apart from silver, selenium is also reported to have excellent antimicrobial property. Chemically synthesized SeNPs are reported to inhibit bacterial growth and biofilm formation and prevent bacterial colonization on orthopedic prostheses [1, 17, 18, 29]. The reason behind antibacterial activity could be oxidative stress [30]. Chudobova et. al. also showed that the antibacterial activity of NPs is size dependent, smaller sized SeNP (50-100 nm) have more antibacterial activity compare to bigger sized silver NP (200-300 nm) [29]. Most of the earlier studies were focused on the bactericidal activity of Se and not specifically on bacterial adherence. Since the nosocomial infection is related to bacterial colonization and biofilm formation on medical devices, our work has focused on bacterial adherence and biofilm inhibition properties of biogenic SeNPs. In an earlier report from our lab, Bacillus licheniformis JS2 derived intracellular biogenic SeNPs were extracted, purified, and characterized for their shape, size, and purity with Dynamic Light Scattering, Scanning and Transmission Electron Microscopy, Fourier Transform Infrared Spectroscopy, Energy Dispersive X-ray Spectroscopy, and SDSPAGE/Silver staining. Moreover, these SeNPs were stable under physiological buffered condition and were non-toxic to the non-cancerous human peripheral blood mononuclear cells [19]. Here, we have demonstrated the bacterial adherence inhibiting properties of SeNPs against a most predominant hospital contaminant, S. aureus, on 3 different surfaces. Coating of these NP reduces the bacterial load significantly to more than 60 % on glass and catheter surface, both in PBS and in the nutrient rich TSB medium. It is interesting to note that S. aureus is not able to form micro-colonies on catheter surface in the presence of SeNPs showing the inhibition of the critical steps of bacterial colonization and biofilm formation by SeNPs. Similar results were obtained on the glass surface in confocal microscopy where 10
thick and dense biofilm was observed on the uncoated cell imaging dish and a thin, and sparsely populated population was observed on SeNP coated CID. These results clearly indicate the S. aureus biofilm inhibiting activity of biogenic SeNPs. The green synthesis approach addressed here for selenium nanoparticles production is simple, eco-friendly, cost effective, and the resultant nanoparticles are non toxic and highly stable [19]. However, the limitation with this system is to control NP size and surface properties. Probably because of this we observed uneven coating of NPs on catheter surface in scanning electron microscopy. This could have lead to bacterial entrapment into the bunch of NPs and thus showing more bacterial count. We are expecting lesser bacterial count on smooth and uniform size coating of SeNPs. Antibacterial and anti-adhesion activity against other pathogens is further needs to be investigated.
5.
Conclusion As per our knowledge, this is the first report to show a comparison in the bacterial
adherence inhibition by biogenic selenium nanoparticles on 3 different surfaces. The results clearly demonstrate that B. licheniformis derived non toxic biogenic SeNPs are promising bacterial adherence inhibiting agent against the employed Gram positive Staphylococcus aureus. The activity of SeNPs can be increased when subjected in combination with other antimicrobial agents.
Acknowledgement We are thankful to the Director, Institute of Microbial Technology, a constituent laboratory of Council of Scientific and Industrial Research (CSIR) for providing excellent infrastructure and facility. This work was financially supported by the Department of Biotechnology, Government of India & CSIR. P.S. was provided research fellowship by CSIR.
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References [1] P.A. Tran, T.J. Webster, Selenium nanoparticles inhibit Staphylococcus aureus growth, Int J Nanomedicine, 6 (2011) 1553-1558. [2] R.O. Darouiche, Device-associated infections: a macroproblem that starts with microadherence, Clin Infect Dis, 33 (2001) 1567-1572. [3] G. Reid, Biofilms in infectious disease and on medical devices, Int J Antimicrob Agents, 11 (1999) 223-226; discussion 237-229. [4] M.J. Richards, J.R. Edwards, D.H. Culver, R.P. Gaynes, Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System, Crit Care Med, 27 (1999) 887-892. [5] R.J. McLean, A.A. Hussain, M. Sayer, P.J. Vincent, D.J. Hughes, T.J. Smith, Antibacterial activity of multilayer silver-copper surface films on catheter material, Can J Microbiol, 39 (1993) 895-899. [6] R.R. Muder, C. Brennen, J.D. Rihs, M.M. Wagener, A. Obman, J.E. Stout, V.L. Yu, Isolation of Staphylococcus aureus from the urinary tract: association of isolation with symptomatic urinary tract infection and subsequent staphylococcal bacteremia, Clin Infect Dis, 42 (2006) 46-50. [7] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections, Science, 284 (1999) 1318-1322. [8] P.S. Stewart, J.W. Costerton, Antibiotic resistance of bacteria in biofilms, Lancet, 358 (2001) 135-138. [9] L.R. Martinez, G. Han, M. Chacko, M.R. Mihu, M. Jacobson, P. Gialanella, A.J. Friedman, J.D. Nosanchuk, J.M. Friedman, Antimicrobial and healing efficacy of sustained release nitric oxide nanoparticles against Staphylococcus aureus skin infection, J Invest Dermatol, 129 (2009) 2463-2469. [10] J.W. Costerton, K.J. Cheng, G.G. Geesey, T.I. Ladd, J.C. Nickel, M. Dasgupta, T.J. Marrie, Bacterial biofilms in nature and disease, Annu Rev Microbiol, 41 (1987) 435-464. [11] A.G. Gristina, Biomaterial-centered infection: microbial adhesion versus tissue integration, Science, 237 (1987) 1588-1595. [12] H. Liedberg, P. Ekman, T. Lundeberg, Pseudomonas aeruginosa: adherence to and growth on different urinary catheter coatings, Int Urol Nephrol, 22 (1990) 487-492. [13] J.R. Johnson, P.L. Roberts, R.J. Olsen, K.A. Moyer, W.E. Stamm, Prevention of catheter-associated urinary tract infection with a silver oxide-coated urinary catheter: clinical and microbiologic correlates, J Infect Dis, 162 (1990) 1145-1150. 12
[14] A.J. Schaeffer, K.O. Story, S.M. Johnson, Effect of silver oxide/trichloroisocyanuric acid antimicrobial urinary drainage system on catheter-associated bacteriuria, J Urol, 139 (1988) 69-73. [15] H. Kucukbay, R. Durmaz, E. Orhan, S. Gunal, Synthesis, antibacterial and antifungal activities of electron-rich olefins derived benzimidazole compounds, Farmaco, 58 (2003) 431-437. [16] M. Pietka-Ottlik, H. Wojtowicz-Mlochowska, K. Kolodziejczyk, E. Piasecki, J. Mlochowski, New organoselenium compounds active against pathogenic bacteria, fungi and viruses, Chem Pharm Bull (Tokyo), 56 (2008) 1423-1427. [17] Q. Wang, T.J. Webster, Short communication: inhibiting biofilm formation on paper towels through the use of selenium nanoparticles coatings, Int J Nanomedicine, 8 (2013) 407411. [18] Q. Wang, T.J. Webster, Nanostructured selenium for preventing biofilm formation on polycarbonate medical devices, J Biomed Mater Res A, 100 (2012) 3205-3210. [19] P. Sonkusre, R. Nanduri, P. Gupta, S.S. Cameotra, Improved extraction of intracellular biogenic selenium nanoparticles and their specificity for cancer chemoprevention, J Nanomed Nanotechnol, 5 (2014) 194-202. [20] S. Dhanjal, S.S. Cameotra, Selenite stress elicits physiological adaptations in Bacillus sp. (strain JS-2), J Microbiol Biotechnol, 21 (2011) 1184-1192. [21] P. Gilbert, D.G. Allison, A.J. McBain, Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?, Symp Ser Soc Appl Microbiol, (2002) 98S-110S. [22] T.F. Mah, G.A. O'Toole, Mechanisms of biofilm resistance to antimicrobial agents, Trends Microbiol, 9 (2001) 34-39. [23] R. Singh, P. Ray, A. Das, M. Sharma, Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study, J Med Microbiol, 58 (2009) 1067-1073. [24] G. Kotzar, M. Freas, P. Abel, A. Fleischman, S. Roy, C. Zorman, J.M. Moran, J. Melzak, Evaluation of MEMS materials of construction for implantable medical devices, Biomaterials, 23 (2002) 2737-2750. [25] X. Ding, C. Yang, T.P. Lim, L.Y. Hsu, A.C. Engler, J.L. Hedrick, Y.Y. Yang, Antibacterial and antifouling catheter coatings using surface grafted PEG-b-cationic polycarbonate diblock copolymers, Biomaterials, 33 (2012) 6593-6603. [26] D. Graiver, R.L. Durall, T. Okada, Surface morphology and friction coefficient of various types of Foley catheter, Biomaterials, 14 (1993) 465-469. 13
[27] D. Fraenkel, C. Rickard, P. Thomas, J. Faoagali, N. George, R. Ware, A prospective, randomized trial of rifampicin-minocycline-coated and silver-platinum-carbon-impregnated central venous catheters, Crit Care Med, 34 (2006) 668-675. [28] D.R. Monteiro, L.F. Gorup, A.S. Takamiya, A.C. Ruvollo-Filho, E.R. de Camargo, D.B. Barbosa, The growing importance of materials that prevent microbial adhesion: antimicrobial effect of medical devices containing silver, Int J Antimicrob Agents, 34 (2009) 103-110. [29] D. Chudobova, K. Cihalova, S. Dostalova, B. Ruttkay-Nedecky, M.A. Rodrigo, K. Tmejova, P. Kopel, L. Nejdl, J. Kudr, J. Gumulec, S. Krizkova, J. Kynicky, R. Kizek, V. Adam, Comparison of the effects of silver phosphate and selenium nanoparticles on Staphylococcus aureus growth reveals potential for selenium particles to prevent infection, FEMS Microbiol Lett, 351 (2014) 195-201. [30] L. Yan, Z. Gu, Y. Zhao, Chemical mechanisms of the toxicological properties of nanomaterials: generation of intracellular reactive oxygen species, Chem Asian J, 8 (2013) 2342-2353.
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Figure legends
Figure 1. (a) SeNP coated 96 well flat bottom polystyrene plate. (b) Absorbance of crystal violet stain at 590 nm corresponds to the number of bacteria present. Significant reduction (*p < 0.05) in bacterial adherence was observed between the control and SeNP (0.5 mg Se/ml) coated surface.
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Figure 2. Confocal microscopy was performed at 100 X oil immersion with 488 nm argon laser. Staining of bacteria with SYTO 9- nucleic acid stain, showing the (a) accumulation of less bacterial population on the SeNP coated glass surface (b) and dense population on uncoated surface. (c) CFU count showing the significant difference in adhered bacterial cell population of the two sets with *p < 0.05. Experiments were performed in triplicates.
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Figure 3. Three dimensional view of biofilm formed by S. aureus on (a) SeNP coated (b) and uncoated glass surface after 3 days of growth in TSB medium. Cells were stained with SYTO 9 and Propidium Iodide for viable and dead cell discrimination. Confocal microscopy was performed at 100 X oil immersion with 488 and 514 nm lasers. 17
Figure 4. SEM images of SeNP coated and uncoated catheter sections incubated with 0.4 OD600 S. aureus culture at 4oC for 24 h. (a) Images showing very few adhered bacteria, marked in red (rest is the SeNPs), on SeNP coated catheter compare to (b) uncoated catheter. (c) CFU count showing the significant difference in adhered bacterial cell population in these two experiment sets with *p < 0.05. Experiments were performed in triplicates.
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Figure 5. SEM images of SeNP coated and uncoated catheter sections incubated at 37oC with 0.6 OD600 S. aureus culture in TSB medium for 72 h. (a) Images showing few isolated, adhered bacteria, indicated by arrows, on SeNP coated catheter whereas, (b) more bacteria with microcolonies were observed on uncoated catheter. These microcolonies will act as precursor for biofilm formation. (c) CFU count confirms the SEM results showing significant difference in adhered cell population between the two sets with *p < 0.05. Experiments were performed in triplicates.
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