Silver Inhibits the Biofilm Formation of Pseudomonas aeruginosa
Author(s)
Bipin Kumar Sharma1,
Animesh Saha1,
Lovely Rahaman1,
Surajit Bhattacharjee2,
Prosun Tribedi1*
Affiliation(s)
1 Department of Microbiology, Tripura University, Suryamaninagar, Tripura, India. 2 Department of Molecular Biology and Bioinformatics, Tripura University, Suryamaninagar, Tripura, India.
1 Department of Microbiology, Tripura University, Suryamaninagar, Tripura, India. 2 Department of Molecular Biology and Bioinformatics, Tripura University, Suryamaninagar, Tripura, India.
ABSTRACT
Biofilm is the assemblage of microbial cells that are irreversibly associated with biotic and abiotic surfaces and is usually enclosed in the self secreted extracellular polymeric substances (EPS). The presence of EPS in biofilm makes the microbial population resistance against antibiotics and other drugs. Biofilms are considered as a serious challenge to pharmaceutical industries because most of the microbial diseases are now associated with biofilm. In this context, we have addressed the biofilm potentialities of Pseudomonas aeruginosa, which has been found to be associated with several deadly diseases including septicemia, urinary tract infections, and gastrointestinal infections, and wherein biofilm plays a crucial role in pathogenesis. Since silver had been used globally for a long time for treating a wide range of illnesses from burn wounds, typhoid, and anthrax to bacterial conjunctivitis in newborns, but its antibiofilm activity is still unknown. Thus, in this current study, we have tried to examine the antibiofilm potentiality of silver against the biofilm of Pseudomonas aeruginosa. Our result showed that silver exhibited considerable antimicrobial property against Pseudomonas aeruginosa where the minimum inhibitory concentration (MIC) was found at 25 μg/ml. Biofilm inhibition by silver against Pseudomonas aeruginosa was then evaluated by crystal violet (CV) staining, estimation of total biofilm protein and microscopy based microbial adherence test using the sub MIC doses of silver. The results showed that all the tested sub MIC doses of silver exhibited considerable antibiofilm activity against P. aeruginosa, wherein the maximum biofilm attenuation was showed by a silver concentration of 20 μg/ml. We also observed that all these sub MIC doses of silver neither interfere with the growth cycle of the bacteria nor affect the cell viability but only attenuates biofilm formation property of the bacteria. The current study deciphers a new axis in biofilm biology where a metal like silver can inhibit the formation of biofilm markedly. Thus, the knowledge gathered in this study may help the pharmaceutical sector to design combinatorial drug where silver could be an important partner to reduce the load of pathogenesity caused by biofilm.
Biofilm is the assemblage of microbial cells that are irreversibly associated with biotic and abiotic surfaces and is usually enclosed in the self secreted extracellular polymeric substances (EPS). The presence of EPS in biofilm makes the microbial population resistance against antibiotics and other drugs. Biofilms are considered as a serious challenge to pharmaceutical industries because most of the microbial diseases are now associated with biofilm. In this context, we have addressed the biofilm potentialities of Pseudomonas aeruginosa, which has been found to be associated with several deadly diseases including septicemia, urinary tract infections, and gastrointestinal infections, and wherein biofilm plays a crucial role in pathogenesis. Since silver had been used globally for a long time for treating a wide range of illnesses from burn wounds, typhoid, and anthrax to bacterial conjunctivitis in newborns, but its antibiofilm activity is still unknown. Thus, in this current study, we have tried to examine the antibiofilm potentiality of silver against the biofilm of Pseudomonas aeruginosa. Our result showed that silver exhibited considerable antimicrobial property against Pseudomonas aeruginosa where the minimum inhibitory concentration (MIC) was found at 25 μg/ml. Biofilm inhibition by silver against Pseudomonas aeruginosa was then evaluated by crystal violet (CV) staining, estimation of total biofilm protein and microscopy based microbial adherence test using the sub MIC doses of silver. The results showed that all the tested sub MIC doses of silver exhibited considerable antibiofilm activity against P. aeruginosa, wherein the maximum biofilm attenuation was showed by a silver concentration of 20 μg/ml. We also observed that all these sub MIC doses of silver neither interfere with the growth cycle of the bacteria nor affect the cell viability but only attenuates biofilm formation property of the bacteria. The current study deciphers a new axis in biofilm biology where a metal like silver can inhibit the formation of biofilm markedly. Thus, the knowledge gathered in this study may help the pharmaceutical sector to design combinatorial drug where silver could be an important partner to reduce the load of pathogenesity caused by biofilm.
Cite this paper
Sharma, B. , Saha, A. , Rahaman, L. , Bhattacharjee, S. and Tribedi, P. (2015) Silver Inhibits the Biofilm Formation of Pseudomonas aeruginosa. Advances in Microbiology, 5, 677-685. doi: 10.4236/aim.2015.510070.
Sharma, B. , Saha, A. , Rahaman, L. , Bhattacharjee, S. and Tribedi, P. (2015) Silver Inhibits the Biofilm Formation of Pseudomonas aeruginosa. Advances in Microbiology, 5, 677-685. doi: 10.4236/aim.2015.510070.
References
[1] Ojha, A.K., Baughn, A.D., Sambandan, D., Hsu, T., Trivelli, X., Guererdal, Y., Alahari, A., Kremer, L., Jacobs Jr., W.R. and Hattful, D.F. (2008) Growth of Mycobacterium tuberculosis Biofilms Containing Free Mycolic Acids and Harbouring Drug-Tolerant Bacteria. Molecular Microbiology, 69, 164-174. http://dx.doi.org/10.1111/j.1365-2958.2008.06274.x [2] Johnson, L.R. (2008) Micro Colony and Biofilm Formation as a Survival Strategy for Bacteria. Journal of Theoretical Biology, 251, 24-34. http://dx.doi.org/10.1016/j.jtbi.2007.10.039 [3] Namasivayam, S.K.R., Preethi, M., Bharani, A.R.S., Robin, G. and Latha, B. (2012) Biofilm Inhibitory Effect of Silver Nanoparticles Coated Catheter against Staphylococcus aureus and Evaluation of Its Synergistic Effects with Antibiotics. International Journal of Biological & Pharmaceutical Research, 3, 259-265. [4] Hall-Stoodley, L. and Stoodley, P. (2005) Biofilm Formation and Dispersal and the Transmission of Human Pathogens. Trends in Microbiology, 13, 7-10. http://dx.doi.org/10.1016/j.tim.2004.11.004 [5] Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R., Teitzel, G.M., Lory, S. and Greenberg, E.P. (2001) Gene expression in Pseudomonas aeruginosa Biofilms. Nature, 413, 860-864. http://dx.doi.org/10.1038/35101627 [6] Gayvallet-Montredon, N., Sauvestre, C., Bergeret, M., Gendrel, D. and Raymond, J. (1998) Bacteriologic Surveillance of Nosocomial Septicemia and Bactiremia in a Pediatric Hospital. Archives de Pédiatrie, 5, 1216-1220. http://dx.doi.org/10.1016/S0929-693X(98)81237-2 [7] Crossley, K.B., Jefferson, K.K., Archer, G.L. and Fowler, V.G. (2009) Staphylococci in Human Disease. 2nd Edition, Blackwell Publishing, Oxford. http://dx.doi.org/10.1002/9781444308464 [8] Yakandawala, N., Gawande, P.V., LoVetri, K. and Madhyastha, S. (2007) Effect of Ovotransferrin, Protamine Sulfate and EDTA Combination on Biofilm Formation by Catheter-Associated Bacteria. Journal of Applied Microbiology, 102, 722-727. http://dx.doi.org/10.1111/j.1365-2672.2006.03129.x [9] Cortes, M.E., Consuegra, J. and Sinisterra, R. (2011) Biofilm Formation, Control and Novel Strategies for Eradication. Science against Microbial Pathogens: Communicating Current Research and Technological Advances, 2, 896-905. [10] Lewis, K. (2001) Riddle of Biofilm Resistance. Antimicrobial Agents and Chemotherapy, 45, 999-1007. http://dx.doi.org/10.1128/AAC.45.4.999-1007.2001 [11] Goossens, H. (2003) Susceptibility of Multi-Drug-Resistant Pseudomonas aeruginosa in Intensive Care Units: Results from the European MYSTIC Study Group. Clinical Microbiology and Infection, 9, 980-983. http://dx.doi.org/10.1046/j.1469-0691.2003.00690.x [12] Wagner, V.E. and Iglewski, B.H. (2008) P. aeruginosa Biofilms in CF Infection. Clinical Reviews in Allergy & Immunology, 35, 124-134. http://dx.doi.org/10.1007/s12016-008-8079-9 [13] Magner, L.N. (1992) Hippocrates and the Hippocratic Tradition: A History of Medicine. Marcel Dekker, Inc., New York. [14] Fung, M.C. and Bowen, D.L. (1996) Silver Products for Medical Indications: Risk-Benefit Assessment. Clinical Toxicology, 34, 119-126. http://dx.doi.org/10.3109/15563659609020246 [15] Rai, M., Yadav, A. and Gade, A. (2009) Silver Nanoparticles as a New Generation of Antimicrobials. Biotechnology Advances, 27, 76-83. http://dx.doi.org/10.1016/j.biotechadv.2008.09.002 [16] Klueh, U., Wagner, V., Kelly, S., Johnson, A. and Bryers, J.D. (2000) Efficacy of Silver-Coated Fabric to Prevent Bacterial Colonization and Subsequent Device-Based Biofilm Formation. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 53, 621-631. http://dx.doi.org/10.1002/1097-4636(2000)53:6<621::AID-JBM2>3.0.CO;2-Q [17] Davies, R.L. and Etris, S.F. (1997) The Development and Functions of Silver in Water Purification and Disease Control. Catalysis Today, 36, 107-114. http://dx.doi.org/10.1016/S0920-5861(96)00203-9 [18] Yamanaka, M., Hara, K. and Kudo, J. (2005) Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis. Applied and Environmental Microbiology, 71, 7589-7593. http://dx.doi.org/10.1128/AEM.71.11.7589-7593.2005 [19] Charles, C. (1974) Current Techniques for Antibiotic Susceptibility Testing. Thomas Publisher, Springfield. [20] Mukherjee, K., Tribedi, P., Mukhopadhyay, B. and Sil, A.K. (2013) Antibacterial Activity of Long-Chain Fatty Alcohols against Mycobacteria. FEMS Microbiology Letters, 338, 177-183. http://dx.doi.org/10.1111/1574-6968.12043 [21] Gurunathan, S., Han, J.W., Kwon, D.N. and Kim, J.H. (2014) Enhanced Antibacterial and Anti-Biofilm Activities of Silver Nanoparticles against Gram-Negative and Gram-Positive Bacteria. Nanoscale Research Letters, 9, 1-17. http://dx.doi.org/10.1186/1556-276X-9-373 [22] Tribedi, P. and Sil, A.K. (2013) Low-Density Polyethylene Degradation by Pseudomonas sp. AKS2 Biofilm. Environmental Science and Pollution Research, 20, 4146-4153. http://dx.doi.org/10.1007/s11356-012-1378-y [23] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein Measurement with the Folin Phenol Reagent. The Journal of Biological Chemistry, 193, 265-275. [24] Donlan, R.M. (2002) Biofilms: Microbial Life on Surfaces. Emerging Infectious Diseases, 8, 881-890. http://dx.doi.org/10.3201/eid0809.020063 [25] Vandeputte, O.M. (2013) Endemic Malagasy Dalbergia Species Inhibit Quorum Sensing in Pseudomonas aeruginosa PAO1. Microbiology, 159, 924-938. http://dx.doi.org/10.1099/mic.0.064378-0 [26] Hall-Stoodley, L. and Stoodley, P. (2009) Evolving Concepts in Biofilm Infections. Cellular Microbiology, 11, 1034-1043. http://dx.doi.org/10.1111/j.1462-5822.2009.01323.x [27] Flemming, H.C. and Wingender, J. (2010) The Biofilm Matrix. Nature Reviews Microbiology, 8, 623-633. http://dx.doi.org/10.1038/nrmicro2415 [28] Bjarnsholt, T., Ciofu, O., Molin, S., Givskov, M. and Hoiby, N. (2013) Applying Insights from Biofilm Biology to Drug Development—Can a New Approach Be Developed? Nature Reviews Drug Discovery, 12, 791-808. http://dx.doi.org/10.1038/nrd4000 [29] Balasubramanian, V., Natarajan, K., Hemambika, B., Ramesh, N., Sumathi, C.S., Kottaimuthu, R. and Rajash, K.V. (2010) High-Density Polyethylene (HDPE)-Degrading Potential Bacteria from Marine Ecosystem of Gulf of Mannar, India. Letters in Applied Microbiology, 51, 205-211. http://dx.doi.org/10.1111/j.1472-765x.2010.02883.x [30] Zhang, H.Q., Zhao, Y., He, X. and Gao, P. (2010) A Novel Approach for Assessing the Susceptibility of Escherichia coli to Antibiotics. Science China Life Sciences, 53, 1346-1355. http://dx.doi.org/10.1007/s11427-010-4087-0 [31] Domenico, P., Baldassarri, L., Schoch, P.E., Kaehler, K., Sasatsu, M. and Cunha, B.A. (2001) Activities of Bismuth Thiols against Staphylococci and Staphylococcal Biofilms. Antimicrobial Agents and Chemotherapy, 45, 1417-1421. http://dx.doi.org/10.1128/AAC.45.5.1417-1421.2001 [32] Khalid, A.Q., AlJohny, B.O. and Wainwright, M. (2014) Antibacterial Effects of Pure Metals on Clinically Important Bacteria Growing in Planktonic Cultures and Biofilms. African Journal of Microbiology Research, 8, 1080-1088. http://dx.doi.org/10.5897/AJMR2013.5893 [33] Hongyan, M., Darmawan, E.T., Zhang, M., Zhange, L. and Bryersa, J.D. (2013) Development of a Poly(ether urethane) System for the Controlled Release of Two Novel Anti-Biofilm Agents Based on Gallium or Zinc and Its Efficacy to Prevent Bacterial Biofilm Formation. Journal of Control Release, 172, 1016. [34] Musk, D.J., Banko, D.A. and Hergenrother, P.J. (2005) Iron Salts Perturb Biofilm Formation and Disrupt Existing Biofilms of Pseudomonas aeruginosa. Journal of Chemistry and Biology, 12, 789-796. http://dx.doi.org/10.1016/j.chembiol.2005.05.007 [35] Jung, W.K., Koo, H.C., Kim, K.W., Shin, S., Kim, S.H. and Park, Y.H. (2008) Antibacterial Activity and Mechanism of Action of the Silver Ion in Staphylococcus aureus and Escherichia coli. Applied and Environmental Microbiology, 74, 2171-2178. http://dx.doi.org/10.1128/AEM.02001-07
[1] Ojha, A.K., Baughn, A.D., Sambandan, D., Hsu, T., Trivelli, X., Guererdal, Y., Alahari, A., Kremer, L., Jacobs Jr., W.R. and Hattful, D.F. (2008) Growth of Mycobacterium tuberculosis Biofilms Containing Free Mycolic Acids and Harbouring Drug-Tolerant Bacteria. Molecular Microbiology, 69, 164-174. http://dx.doi.org/10.1111/j.1365-2958.2008.06274.x [2] Johnson, L.R. (2008) Micro Colony and Biofilm Formation as a Survival Strategy for Bacteria. Journal of Theoretical Biology, 251, 24-34. http://dx.doi.org/10.1016/j.jtbi.2007.10.039 [3] Namasivayam, S.K.R., Preethi, M., Bharani, A.R.S., Robin, G. and Latha, B. (2012) Biofilm Inhibitory Effect of Silver Nanoparticles Coated Catheter against Staphylococcus aureus and Evaluation of Its Synergistic Effects with Antibiotics. International Journal of Biological & Pharmaceutical Research, 3, 259-265. [4] Hall-Stoodley, L. and Stoodley, P. (2005) Biofilm Formation and Dispersal and the Transmission of Human Pathogens. Trends in Microbiology, 13, 7-10. http://dx.doi.org/10.1016/j.tim.2004.11.004 [5] Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R., Teitzel, G.M., Lory, S. and Greenberg, E.P. (2001) Gene expression in Pseudomonas aeruginosa Biofilms. Nature, 413, 860-864. http://dx.doi.org/10.1038/35101627 [6] Gayvallet-Montredon, N., Sauvestre, C., Bergeret, M., Gendrel, D. and Raymond, J. (1998) Bacteriologic Surveillance of Nosocomial Septicemia and Bactiremia in a Pediatric Hospital. Archives de Pédiatrie, 5, 1216-1220. http://dx.doi.org/10.1016/S0929-693X(98)81237-2 [7] Crossley, K.B., Jefferson, K.K., Archer, G.L. and Fowler, V.G. (2009) Staphylococci in Human Disease. 2nd Edition, Blackwell Publishing, Oxford. http://dx.doi.org/10.1002/9781444308464 [8] Yakandawala, N., Gawande, P.V., LoVetri, K. and Madhyastha, S. (2007) Effect of Ovotransferrin, Protamine Sulfate and EDTA Combination on Biofilm Formation by Catheter-Associated Bacteria. Journal of Applied Microbiology, 102, 722-727. http://dx.doi.org/10.1111/j.1365-2672.2006.03129.x [9] Cortes, M.E., Consuegra, J. and Sinisterra, R. (2011) Biofilm Formation, Control and Novel Strategies for Eradication. Science against Microbial Pathogens: Communicating Current Research and Technological Advances, 2, 896-905. [10] Lewis, K. (2001) Riddle of Biofilm Resistance. Antimicrobial Agents and Chemotherapy, 45, 999-1007. http://dx.doi.org/10.1128/AAC.45.4.999-1007.2001 [11] Goossens, H. (2003) Susceptibility of Multi-Drug-Resistant Pseudomonas aeruginosa in Intensive Care Units: Results from the European MYSTIC Study Group. Clinical Microbiology and Infection, 9, 980-983. http://dx.doi.org/10.1046/j.1469-0691.2003.00690.x [12] Wagner, V.E. and Iglewski, B.H. (2008) P. aeruginosa Biofilms in CF Infection. Clinical Reviews in Allergy & Immunology, 35, 124-134. http://dx.doi.org/10.1007/s12016-008-8079-9 [13] Magner, L.N. (1992) Hippocrates and the Hippocratic Tradition: A History of Medicine. Marcel Dekker, Inc., New York. [14] Fung, M.C. and Bowen, D.L. (1996) Silver Products for Medical Indications: Risk-Benefit Assessment. Clinical Toxicology, 34, 119-126. http://dx.doi.org/10.3109/15563659609020246 [15] Rai, M., Yadav, A. and Gade, A. (2009) Silver Nanoparticles as a New Generation of Antimicrobials. Biotechnology Advances, 27, 76-83. http://dx.doi.org/10.1016/j.biotechadv.2008.09.002 [16] Klueh, U., Wagner, V., Kelly, S., Johnson, A. and Bryers, J.D. (2000) Efficacy of Silver-Coated Fabric to Prevent Bacterial Colonization and Subsequent Device-Based Biofilm Formation. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 53, 621-631. http://dx.doi.org/10.1002/1097-4636(2000)53:6<621::AID-JBM2>3.0.CO;2-Q [17] Davies, R.L. and Etris, S.F. (1997) The Development and Functions of Silver in Water Purification and Disease Control. Catalysis Today, 36, 107-114. http://dx.doi.org/10.1016/S0920-5861(96)00203-9 [18] Yamanaka, M., Hara, K. and Kudo, J. (2005) Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis. Applied and Environmental Microbiology, 71, 7589-7593. http://dx.doi.org/10.1128/AEM.71.11.7589-7593.2005 [19] Charles, C. (1974) Current Techniques for Antibiotic Susceptibility Testing. Thomas Publisher, Springfield. [20] Mukherjee, K., Tribedi, P., Mukhopadhyay, B. and Sil, A.K. (2013) Antibacterial Activity of Long-Chain Fatty Alcohols against Mycobacteria. FEMS Microbiology Letters, 338, 177-183. http://dx.doi.org/10.1111/1574-6968.12043 [21] Gurunathan, S., Han, J.W., Kwon, D.N. and Kim, J.H. (2014) Enhanced Antibacterial and Anti-Biofilm Activities of Silver Nanoparticles against Gram-Negative and Gram-Positive Bacteria. Nanoscale Research Letters, 9, 1-17. http://dx.doi.org/10.1186/1556-276X-9-373 [22] Tribedi, P. and Sil, A.K. (2013) Low-Density Polyethylene Degradation by Pseudomonas sp. AKS2 Biofilm. Environmental Science and Pollution Research, 20, 4146-4153. http://dx.doi.org/10.1007/s11356-012-1378-y [23] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein Measurement with the Folin Phenol Reagent. The Journal of Biological Chemistry, 193, 265-275. [24] Donlan, R.M. (2002) Biofilms: Microbial Life on Surfaces. Emerging Infectious Diseases, 8, 881-890. http://dx.doi.org/10.3201/eid0809.020063 [25] Vandeputte, O.M. (2013) Endemic Malagasy Dalbergia Species Inhibit Quorum Sensing in Pseudomonas aeruginosa PAO1. Microbiology, 159, 924-938. http://dx.doi.org/10.1099/mic.0.064378-0 [26] Hall-Stoodley, L. and Stoodley, P. (2009) Evolving Concepts in Biofilm Infections. Cellular Microbiology, 11, 1034-1043. http://dx.doi.org/10.1111/j.1462-5822.2009.01323.x [27] Flemming, H.C. and Wingender, J. (2010) The Biofilm Matrix. Nature Reviews Microbiology, 8, 623-633. http://dx.doi.org/10.1038/nrmicro2415 [28] Bjarnsholt, T., Ciofu, O., Molin, S., Givskov, M. and Hoiby, N. (2013) Applying Insights from Biofilm Biology to Drug Development—Can a New Approach Be Developed? Nature Reviews Drug Discovery, 12, 791-808. http://dx.doi.org/10.1038/nrd4000 [29] Balasubramanian, V., Natarajan, K., Hemambika, B., Ramesh, N., Sumathi, C.S., Kottaimuthu, R. and Rajash, K.V. (2010) High-Density Polyethylene (HDPE)-Degrading Potential Bacteria from Marine Ecosystem of Gulf of Mannar, India. Letters in Applied Microbiology, 51, 205-211. http://dx.doi.org/10.1111/j.1472-765x.2010.02883.x [30] Zhang, H.Q., Zhao, Y., He, X. and Gao, P. (2010) A Novel Approach for Assessing the Susceptibility of Escherichia coli to Antibiotics. Science China Life Sciences, 53, 1346-1355. http://dx.doi.org/10.1007/s11427-010-4087-0 [31] Domenico, P., Baldassarri, L., Schoch, P.E., Kaehler, K., Sasatsu, M. and Cunha, B.A. (2001) Activities of Bismuth Thiols against Staphylococci and Staphylococcal Biofilms. Antimicrobial Agents and Chemotherapy, 45, 1417-1421. http://dx.doi.org/10.1128/AAC.45.5.1417-1421.2001 [32] Khalid, A.Q., AlJohny, B.O. and Wainwright, M. (2014) Antibacterial Effects of Pure Metals on Clinically Important Bacteria Growing in Planktonic Cultures and Biofilms. African Journal of Microbiology Research, 8, 1080-1088. http://dx.doi.org/10.5897/AJMR2013.5893 [33] Hongyan, M., Darmawan, E.T., Zhang, M., Zhange, L. and Bryersa, J.D. (2013) Development of a Poly(ether urethane) System for the Controlled Release of Two Novel Anti-Biofilm Agents Based on Gallium or Zinc and Its Efficacy to Prevent Bacterial Biofilm Formation. Journal of Control Release, 172, 1016. [34] Musk, D.J., Banko, D.A. and Hergenrother, P.J. (2005) Iron Salts Perturb Biofilm Formation and Disrupt Existing Biofilms of Pseudomonas aeruginosa. Journal of Chemistry and Biology, 12, 789-796. http://dx.doi.org/10.1016/j.chembiol.2005.05.007 [35] Jung, W.K., Koo, H.C., Kim, K.W., Shin, S., Kim, S.H. and Park, Y.H. (2008) Antibacterial Activity and Mechanism of Action of the Silver Ion in Staphylococcus aureus and Escherichia coli. Applied and Environmental Microbiology, 74, 2171-2178. http://dx.doi.org/10.1128/AEM.02001-07