SCANNING VOL. 36, 551–553 (2014) © 2014 Wiley Periodicals, Inc.
Nanoscale Investigation on Pseudomonas aeruginosa Biofilm Formed on Porous Silicon using Atomic Force Microscopy ASHWIN KANNAN, SUBBALAKSHMI LATHA KARUMANCHI, VINATHA KRISHNA, KOTHAI THIRUVENGADAM, SUBRAMANIAM RAMALINGAM, AND PENNATHUR GAUTAM Centre for Biotechnology, Anna University, Chennai, India
Summary: Colonization of surfaces by bacterial cells results in the formation of biofilms. There is a need to study the factors that are important for formation of biofilms since biofilms have been implicated in the failure of semiconductor devices and implants. In the present study, the adhesion force of biofilms (formed by Pseudomonas aeruginosa) on porous silicon substrates of varying surface roughness was quantified using atomic force microscopy (AFM). The experiments were carried out to quantify the effect of surface roughness on the adhesion force of biofilm. The results show that the adhesion force increased from 1.5 0.5 to 13.2 0.9 nN with increase in the surface roughness of silicon substrate. The results suggest that the adhesion force of biofilm is affected by surface roughness of substrate. SCANNING 36:551–553, 2014. © 2014 Wiley Periodicals, Inc. Key words: Pseudomonas aeruginosa, biofilm, atomic force microscopy, porous silicon, surface roughness Bacterial attachment to surfaces is affected by factors like surface topography, temperature, surface characteristics of the material and cellular characteristics of the bacteria (Katsikogianni and Missirlis 2004). Colonization of surfaces by bacterial cells leads to the formation of biofilms. Biofilms are aggregates of microorganisms embedded in a matrix of extracellular polymeric material (Colvin et al. 2011). Biofilm formation is a multistep process initiated by bacterial adhesion to solid Contract grant sponsor: Department of Biotechnology.Contract grant sponsor: Department of Science & Technology. Conflict of Interest: None. Address for reprints: P. Gautam, Centre for Biotechnology, Anna University, Chennai, 25, India E-mail: [email protected] Received 19 March 2014; Revised 20 April 2014; Accepted with revision 5 May 2014 DOI: 10.1002/sca.21148 Published online 10 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
surfaces (Dunne 2002). Colonization of surfaces by bacteria promotes the formation of micro-colonies resulting in formation of biofilms. This is followed by their growth and maturation (Dunne 2002). Biofilms thus formed, help in immobilization of the cells and also function as a protective barrier against antibiotics (Colvin et al. 2011). Formation of biofilms has been the main cause for failure of implants since the bacterial cells within the biofilm matrix exibit enhanced tolerance to the effect of antimicrobial agents. Formation of biofilms has also been reported to cause heavy losses in industries due to biocorrosion (Katsikogianni and Missirlis 2004). They have also been implicated in water contamination leading to a multitude of diseases. Hence there is a need to study the factors that are crucial for biofilm formation. The adherence of hippocampal cells on silicon wafers with nano-scale morphology was studied using atomic force microscopy (AFM) and it was found that the frictional force on the boundary around the cells increased due to accumulation of proteins (Ma et al. 2005). Fan et al. (2002) studied the effect of surface roughness on the adherence and viability of central neural cells and reported that cell adhesion and viability were significantly affected by surface roughness (on silicon wafers). The effect of nanostructured surfaces (on bacterial adhesion and subsequent biofilm formation) has been studied using confocal microscopy (Singh et al. 2011). Singh et al. (2011) reported that bacterial adhesion and biofilm formation were enhanced on surfaces with roughness upto 20 nm and that a further increase in surface roughness caused a decrease in bacterial adhesion and inhibited biofilm formation. Oh et al. (2009) studied the formation of biofilms on different substrates (aluminium, steel, rubber, and polypropylene) using AFM and suggested that surface roughness (of the substrates) could have an effect on biofilm adhesion force (or tip-biofilm interaction force). The main drawback of this work was that surface roughness parameters (characterizing surface roughness) for each of the substrates were not evaluated. The other drawback was that substrates
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varying in both surface topography and surface (chemical) characteristics were used for the study. Hence, the true relationship between surface roughness (of the substrate) and biofilm adhesion force was not evaluated. In the present study, AFM was used to study the effect of surface roughness (of silicon substrate) on the biofilm adhesion force. This is of importance since biofilm formation on silicon based semiconductor devices is one of the major concerns in the semiconductor industry. Polished silicon wafers used in this study were n-type (100) and were procured from Sigma–Aldrich, USA. Silicon substrates were used because nanopatterned surfaces of varying surface roughness can be easily fabricated by electrochemical etching of polished silicon substrates. In addition to this, it is also known that bacteria grow on silicon substrates used in electronics industry and cause biofouling (Scheuerman et al. ’98). Porous silicon substrates (PSS) were fabricated by etching polished wafers using a Potentiostat (CHI instruments, model 1140A) (Sailor 2011). PSS were then rinsed with pentane and then thermally oxidized to passivate the PSS (Jarvis et al. 2008). The surface topography of PSS was then resolved (in intermittent contact mode using JPK Nanowizard II) over an area of 5 mm 5 mm (Figs. S1–S7). The parameters Ra and Rq were then evaluated for the PSS (Table I). The organism Pseudomonas aeruginosa (MTCC 2297) was chosen for our studies. P. aeruginosa was chosen since it is a model organism used in biofilm research. P. aeruginosa culture grown in nutrient broth (2% (v/v) of 0.8 A620) was used to inoculate the biofilm production media. The biofilm production media consisted of minimal media with 0.5% (w/w) magnesium palmitate. Magnesium palmitate was used since bacterial cells use it as a carbon source. Magnesium palmitate was prepared according to the procedure given by Quraishi et al. (2012). Biofilm samples formed on the PSS were rinsed with phosphate buffer saline (pH 7.2) solution (Oh et al. 2009). AFM images of biofilm
TABLE I Surface roughness of the porous silicon substrates PSS 1 2 3 4 5 6
Ra (nm)
Rq (nm)
Adhesion force (nN)
1.3 8.6 17.8 19.7 26.3 55.2
1.7 10.9 23.1 25.8 32 69.9
1.5 0.5 2.8 1.2 5.0 0.6 7.40 1.7 11.40 0.8 13.20 0.9
Ra, average deviation from profile centre.Rq, root mean square deviation.
formed on PSS were resolved in contact mode using Mikromasch CSC38 probes (Figs. S8–S13). All (adhesion) force measurements were conducted using Mikromasch CSC38 probes and experiments were performed at a relative humidity of 30%. The force constant of the probes used were 0.03 Nm 1. The probes were calibrated before use. Force-distance curves (FDC) were generated to study the effect of surface roughness on the biofilm adhesion force. A FDC provides helps in evaluating the interaction between the probe and biofilm. A total of 64 FDC were generated. The biofilm adhesion force on each of the PSS was then calculated. The adhesion force was found to vary from 1.5 0.5 nN (for biofilm sample on PSS1) to 13.20 0.9 nN (for biofilm sample on PSS6). A plot of adhesion force against surface roughness parameters (Ra and Rq) revealed the effect of these parameters on the adhesion force (Fig. 1). The parameter Ra measures the arithmetic average of deviation about the mean profile line and is the most commonly used surface roughness parameter. This parameter does not differentiate between peaks and valleys. The parameter Rq measures the root mean square average about the mean profile line and is more sensitive to variations in surface topography (than Ra) since the amplitude term is squared. The plot of adhesion force against surface roughness parameters showed that the adhesion force increased with the surface roughness. The observed increase in the adhesion force is due to increased production and accumulation of extracellular polymeric material (Oh et al. 2009; Fang et al. 2000). These results suggest that the adhesion force of biofilm could be reduced by using smooth surfaces in the nanoscale. The results of this study may help in the fabrication of better silicon based devices (since biofilm formation on silicon based semiconductor devices is one of the major concerns in the semiconductor industry) and silicon based implants.
Conclusion
Fig. 1. Plot of adhesion force versus surface roughness parameters Ra and Rq.
The effect of nanometer surface roughness (of substrate) on biofilm adhesiveness was studied using AFM. The results indicate that the adhesion force of biofilm increases with increase in surface roughness.
A. Kannan et al.: Studies on Pseudomonas aeruginosa biofilm
The results suggest that the adhesion force of biofilm could be reduced by using smooth surfaces in the nanoscale. This may help in the fabrication of better silicon based biomaterials and devices.
Acknowledgment The authors would like to thank the Department of Biotechnology and Department of Science & Technology for their continued financial support. The funding agency had no role in the design of experiments, data collection and analysis, decision to publish or preparation of the manuscript.
References Colvin KM, Gordon VD, Murakami K, Borlee BR, Wozniak DJ, Wong GCL, Parsek MR. 2011. The Pel Polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog 7:e1001264. doi: 10.1371/journal.ppat.1001264 Dunne WM. 2002. Bacterial adhesion: seen any good biofilms lately? Clin Microbiol Rev 15:155–166. Fan YW, Cui FZ, Hou SP, et al. 2002. Culture of neural cells on silicon wafers with nano-scale surface topograph. J Neurosci Methods 120:17–23. Fang HHP, Chan KY, Xu LC. 2000. Quantification of bacterial adhesion forces using atomic force microscopy. J Microbiol Methods 40:89–97.
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Jarvis KL, Barnes TJ, Prestidge CA. 2008. Aqueous and thermal oxidation of porous silicon microparticles: implications on molecular interactions. Langmuir 24:14222–14226. Katsikogianni M, Missirlis YF. 2004. Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions. Eur Cell Mater 8:37–57. Ma J, Liu BF, Xu QY, Cui FZ. 2005. AFM study of hippocampal cells cultured on silicon wafers with nano-scale surface topograph. Colloid Surf B Biointerfaces 44:152–157. Oh YJ, Lee NR, Jo W, Jung WK, Lim JS. 2009. Effects of substrates on biofilm formation observed by atomic force microscopy. Ultramicroscopy 109:874–880. Quraishi MA, Kumar V, Singh BN, Singh SK. 2012. Calcium palmitate: a green corrosion inhibitor for steel in concrete environment. J Mater Environ Sci 3:1001–1008. Sailor MJ. 2011. Porous silicon in practice: preparation, characterization and applications. Germany: Wiley-VCH, Weinheim edition. Scheuerman TR, Camper AK, Hamilton A. 1998. Effects of substratum Topography on bacterial adhesion. J Colloid Interface Sci 208:23–33. Singh AV, Vyas V, Patil R, et al. 2011. Quantitative characterization of the influence of the nanoscale morphology of nanostructured surfaces on bacterial adhesion and biofilm formation. PLoS ONE 6:e25029.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
Nanoscale Investigation on Pseudomonas aeruginosa Biofilm Formed on Porous Silicon using Atomic Force Microscopy ASHWIN KANNAN, SUBBALAKSHMI LATHA KARUMANCHI, VINATHA KRISHNA, KOTHAI THIRUVENGADAM, SUBRAMANIAM RAMALINGAM, AND PENNATHUR GAUTAM Centre for Biotechnology, Anna University, Chennai, India
Summary: Colonization of surfaces by bacterial cells results in the formation of biofilms. There is a need to study the factors that are important for formation of biofilms since biofilms have been implicated in the failure of semiconductor devices and implants. In the present study, the adhesion force of biofilms (formed by Pseudomonas aeruginosa) on porous silicon substrates of varying surface roughness was quantified using atomic force microscopy (AFM). The experiments were carried out to quantify the effect of surface roughness on the adhesion force of biofilm. The results show that the adhesion force increased from 1.5 0.5 to 13.2 0.9 nN with increase in the surface roughness of silicon substrate. The results suggest that the adhesion force of biofilm is affected by surface roughness of substrate. SCANNING 36:551–553, 2014. © 2014 Wiley Periodicals, Inc. Key words: Pseudomonas aeruginosa, biofilm, atomic force microscopy, porous silicon, surface roughness Bacterial attachment to surfaces is affected by factors like surface topography, temperature, surface characteristics of the material and cellular characteristics of the bacteria (Katsikogianni and Missirlis 2004). Colonization of surfaces by bacterial cells leads to the formation of biofilms. Biofilms are aggregates of microorganisms embedded in a matrix of extracellular polymeric material (Colvin et al. 2011). Biofilm formation is a multistep process initiated by bacterial adhesion to solid Contract grant sponsor: Department of Biotechnology.Contract grant sponsor: Department of Science & Technology. Conflict of Interest: None. Address for reprints: P. Gautam, Centre for Biotechnology, Anna University, Chennai, 25, India E-mail: [email protected] Received 19 March 2014; Revised 20 April 2014; Accepted with revision 5 May 2014 DOI: 10.1002/sca.21148 Published online 10 July 2014 in Wiley Online Library (wileyonlinelibrary.com).
surfaces (Dunne 2002). Colonization of surfaces by bacteria promotes the formation of micro-colonies resulting in formation of biofilms. This is followed by their growth and maturation (Dunne 2002). Biofilms thus formed, help in immobilization of the cells and also function as a protective barrier against antibiotics (Colvin et al. 2011). Formation of biofilms has been the main cause for failure of implants since the bacterial cells within the biofilm matrix exibit enhanced tolerance to the effect of antimicrobial agents. Formation of biofilms has also been reported to cause heavy losses in industries due to biocorrosion (Katsikogianni and Missirlis 2004). They have also been implicated in water contamination leading to a multitude of diseases. Hence there is a need to study the factors that are crucial for biofilm formation. The adherence of hippocampal cells on silicon wafers with nano-scale morphology was studied using atomic force microscopy (AFM) and it was found that the frictional force on the boundary around the cells increased due to accumulation of proteins (Ma et al. 2005). Fan et al. (2002) studied the effect of surface roughness on the adherence and viability of central neural cells and reported that cell adhesion and viability were significantly affected by surface roughness (on silicon wafers). The effect of nanostructured surfaces (on bacterial adhesion and subsequent biofilm formation) has been studied using confocal microscopy (Singh et al. 2011). Singh et al. (2011) reported that bacterial adhesion and biofilm formation were enhanced on surfaces with roughness upto 20 nm and that a further increase in surface roughness caused a decrease in bacterial adhesion and inhibited biofilm formation. Oh et al. (2009) studied the formation of biofilms on different substrates (aluminium, steel, rubber, and polypropylene) using AFM and suggested that surface roughness (of the substrates) could have an effect on biofilm adhesion force (or tip-biofilm interaction force). The main drawback of this work was that surface roughness parameters (characterizing surface roughness) for each of the substrates were not evaluated. The other drawback was that substrates
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varying in both surface topography and surface (chemical) characteristics were used for the study. Hence, the true relationship between surface roughness (of the substrate) and biofilm adhesion force was not evaluated. In the present study, AFM was used to study the effect of surface roughness (of silicon substrate) on the biofilm adhesion force. This is of importance since biofilm formation on silicon based semiconductor devices is one of the major concerns in the semiconductor industry. Polished silicon wafers used in this study were n-type (100) and were procured from Sigma–Aldrich, USA. Silicon substrates were used because nanopatterned surfaces of varying surface roughness can be easily fabricated by electrochemical etching of polished silicon substrates. In addition to this, it is also known that bacteria grow on silicon substrates used in electronics industry and cause biofouling (Scheuerman et al. ’98). Porous silicon substrates (PSS) were fabricated by etching polished wafers using a Potentiostat (CHI instruments, model 1140A) (Sailor 2011). PSS were then rinsed with pentane and then thermally oxidized to passivate the PSS (Jarvis et al. 2008). The surface topography of PSS was then resolved (in intermittent contact mode using JPK Nanowizard II) over an area of 5 mm 5 mm (Figs. S1–S7). The parameters Ra and Rq were then evaluated for the PSS (Table I). The organism Pseudomonas aeruginosa (MTCC 2297) was chosen for our studies. P. aeruginosa was chosen since it is a model organism used in biofilm research. P. aeruginosa culture grown in nutrient broth (2% (v/v) of 0.8 A620) was used to inoculate the biofilm production media. The biofilm production media consisted of minimal media with 0.5% (w/w) magnesium palmitate. Magnesium palmitate was used since bacterial cells use it as a carbon source. Magnesium palmitate was prepared according to the procedure given by Quraishi et al. (2012). Biofilm samples formed on the PSS were rinsed with phosphate buffer saline (pH 7.2) solution (Oh et al. 2009). AFM images of biofilm
TABLE I Surface roughness of the porous silicon substrates PSS 1 2 3 4 5 6
Ra (nm)
Rq (nm)
Adhesion force (nN)
1.3 8.6 17.8 19.7 26.3 55.2
1.7 10.9 23.1 25.8 32 69.9
1.5 0.5 2.8 1.2 5.0 0.6 7.40 1.7 11.40 0.8 13.20 0.9
Ra, average deviation from profile centre.Rq, root mean square deviation.
formed on PSS were resolved in contact mode using Mikromasch CSC38 probes (Figs. S8–S13). All (adhesion) force measurements were conducted using Mikromasch CSC38 probes and experiments were performed at a relative humidity of 30%. The force constant of the probes used were 0.03 Nm 1. The probes were calibrated before use. Force-distance curves (FDC) were generated to study the effect of surface roughness on the biofilm adhesion force. A FDC provides helps in evaluating the interaction between the probe and biofilm. A total of 64 FDC were generated. The biofilm adhesion force on each of the PSS was then calculated. The adhesion force was found to vary from 1.5 0.5 nN (for biofilm sample on PSS1) to 13.20 0.9 nN (for biofilm sample on PSS6). A plot of adhesion force against surface roughness parameters (Ra and Rq) revealed the effect of these parameters on the adhesion force (Fig. 1). The parameter Ra measures the arithmetic average of deviation about the mean profile line and is the most commonly used surface roughness parameter. This parameter does not differentiate between peaks and valleys. The parameter Rq measures the root mean square average about the mean profile line and is more sensitive to variations in surface topography (than Ra) since the amplitude term is squared. The plot of adhesion force against surface roughness parameters showed that the adhesion force increased with the surface roughness. The observed increase in the adhesion force is due to increased production and accumulation of extracellular polymeric material (Oh et al. 2009; Fang et al. 2000). These results suggest that the adhesion force of biofilm could be reduced by using smooth surfaces in the nanoscale. The results of this study may help in the fabrication of better silicon based devices (since biofilm formation on silicon based semiconductor devices is one of the major concerns in the semiconductor industry) and silicon based implants.
Conclusion
Fig. 1. Plot of adhesion force versus surface roughness parameters Ra and Rq.
The effect of nanometer surface roughness (of substrate) on biofilm adhesiveness was studied using AFM. The results indicate that the adhesion force of biofilm increases with increase in surface roughness.
A. Kannan et al.: Studies on Pseudomonas aeruginosa biofilm
The results suggest that the adhesion force of biofilm could be reduced by using smooth surfaces in the nanoscale. This may help in the fabrication of better silicon based biomaterials and devices.
Acknowledgment The authors would like to thank the Department of Biotechnology and Department of Science & Technology for their continued financial support. The funding agency had no role in the design of experiments, data collection and analysis, decision to publish or preparation of the manuscript.
References Colvin KM, Gordon VD, Murakami K, Borlee BR, Wozniak DJ, Wong GCL, Parsek MR. 2011. The Pel Polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog 7:e1001264. doi: 10.1371/journal.ppat.1001264 Dunne WM. 2002. Bacterial adhesion: seen any good biofilms lately? Clin Microbiol Rev 15:155–166. Fan YW, Cui FZ, Hou SP, et al. 2002. Culture of neural cells on silicon wafers with nano-scale surface topograph. J Neurosci Methods 120:17–23. Fang HHP, Chan KY, Xu LC. 2000. Quantification of bacterial adhesion forces using atomic force microscopy. J Microbiol Methods 40:89–97.
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Jarvis KL, Barnes TJ, Prestidge CA. 2008. Aqueous and thermal oxidation of porous silicon microparticles: implications on molecular interactions. Langmuir 24:14222–14226. Katsikogianni M, Missirlis YF. 2004. Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions. Eur Cell Mater 8:37–57. Ma J, Liu BF, Xu QY, Cui FZ. 2005. AFM study of hippocampal cells cultured on silicon wafers with nano-scale surface topograph. Colloid Surf B Biointerfaces 44:152–157. Oh YJ, Lee NR, Jo W, Jung WK, Lim JS. 2009. Effects of substrates on biofilm formation observed by atomic force microscopy. Ultramicroscopy 109:874–880. Quraishi MA, Kumar V, Singh BN, Singh SK. 2012. Calcium palmitate: a green corrosion inhibitor for steel in concrete environment. J Mater Environ Sci 3:1001–1008. Sailor MJ. 2011. Porous silicon in practice: preparation, characterization and applications. Germany: Wiley-VCH, Weinheim edition. Scheuerman TR, Camper AK, Hamilton A. 1998. Effects of substratum Topography on bacterial adhesion. J Colloid Interface Sci 208:23–33. Singh AV, Vyas V, Patil R, et al. 2011. Quantitative characterization of the influence of the nanoscale morphology of nanostructured surfaces on bacterial adhesion and biofilm formation. PLoS ONE 6:e25029.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
