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Studies on Effect of TiO2 Nanoparticles on Growth and Membrane Permeability of Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis
I. Mohammed Sadiq, N. Chandrasekaran and A. Mukherjee*
Nanobio-Medicine Research Group, School of Bio Sciences & Technology, VIT- University, Vellore-632014, India
Abstract: Metal oxide nanoparticles are known to possess strong antimicrobial properties. Titanium dioxide (titania) nanoparticles have wide range antimicrobial as well biomedical applications. In the present work the antimicrobial properties of Titanium dioxide- anatase nanoparticles were investigated in the concentration range 10-100 ppm using Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa. The average size of the nanoparticles used was measured to be below 25 nm. The concentration dependent growth inhibitory effect of titania nanoparticles on the three bacterial strains were compared based on batch growth kinetic data (by dynamic growth rate). Bacterial sensitivity to nanoparticles was found to vary depending on the microbial species. The mode of antibacterial action was investigated by assaying extracellular proteins, the integrity of the cell membranes and the permeability of the inner membrane (IM) of the microbial strains. The results suggested that titania caused the antibacterial effect through membrane damage mechanisms. FT-IR studies revealed that most of amide-I, amide II groups were absent in the nanoparticles treated cells when compared with that of control, corroborating the cell damage mode of action. The antimicrobial effects of the nanoparticles essentially differed between the microbial strains due to structural differences in the cell membranes.

Keywords: Titanium dioxide (anatase), antimicrobial, batch growth kinetics, Membrane integrity, Inner membrane permeability. 1. INTRODUCTION The emergence of nanoscience and nanotechnology in the last decade presents opportunities for exploring the bactericidal effect of metal nanoparticles. The bactericidal effect of metal nanoparticles has been attributed to their small size and high surface to volume ratio which allows them to interact closely with microbial membranes and is not merely due to the release of metal ions in solution [1]. Metal nanoparticles with bactericidal activity can be immobilized and coated on to surfaces, which may find application in various fields, i.e., medical instruments and devices, water treatment and food processing. Metal nanoparticles have been combined with polymers to form composites for better utilization of their antimicrobial activity. Metal nanoparticles are also useful in various other fields, i.e., catalysis and sensors [2, 3]. However, it is gradually being recognized that nanoparticles may have many undesirable impact on the environment and in the ecosystem [4, 5]. The use of photocatalytic semiconductor oxides emerges as a successful technology to struggle against biological risks. TiO2- Anatase is by far the most widely used photocatalyst, being a wide band-gap (3.2 eV) semiconductor that under UV illumination generates energyrich electron hole pairs able to degrade cell components of microorganisms rendering innocuous products. Bactericidal activity of TiO2/UV reaction and its killing mechanism was also reported by Maness and co-workers and the lipid peroxidation reaction was the first evidence proposed as the underlying mechanism of the death of E. coli [6]. Very few studies have been conducted with Titania in absence of photo-activation effect. Many researchers reported that anatase Titania nanoparticles have higher photo activity than the rutile [7]. Most of the researchers have so far demonstrated the photocatalytic killing mechanism of Titanium dioxide on different microorganism using UVA, UVB, UVC and solar sources of light. The mechanisms by which the cell membranes lose structural integrity in the presence of metal nanoparticles are currently thought to involve lipid peroxidation by reactive oxygen species (ROS) such as superoxide (O2) and hydroxyl radicals (OH), as described in case of eukaryotic cells [8]. Using TiO2 nanoparticles in the presence of UV-radiation and O2, ROS are thought to be generated to the extent to cause sufficient microbial toxicity; in these cases direct contact between nanoparticles and the bacterial membrane appears imperative [9, 10, 11]. The objective of the present study was to find out the variabilities in bactericidal effect of Titania (Anatase) nanoparticles on three representative bacterial strains i.e., E. coli, B. subtilis and P. aeruginosa . Such a comparative study would eventually lead to better utilization of nanoparticles for specific antibacterial applications. The mode of bactericidal action of nano Titania on these three bacterial strains were investigated by measuring extracellular protein content, studying the cell wall integrity, and cell membrane permeability. The structural differences induced by the action of the Titania nanoparticles were detailed by FTIR spectroscopic studies. 2. MATERIALS AND METHODS 2.1. Materials and Bacterial Strains The antibacterial sensitivity tests were carried out with two Gram negative bacteria: E.coli (ATCC 25922), Pseudomonas aeruginosa (NCIM 2200, ATCC 9027) and one Gram positive bacteria: Bacillus subtilis (NCIM 2063, ATCC 6633). These bacterial strains were selected since these organisms are extensively used for antibiotic susceptibility assays, antimicrobial activity assays and sterility testing assays. Strains of these bacteria were abundant in natural environment and easily culturable. E.coli strain was procured from the MTCC Chandigarh India and other two strains were procured from the National Chemical Laboratory (Pune, India). E.coli and Bacillus strains were maintained in Luria Bertani media (Himedia Laboratories Ltd., Mumbai, India), Cetrimide agar was used for sub-culturing of Pseudomonas aeruginosa. Throughout this study, the same nutrient medium (Himedia Laboratories Ltd., Mumbai, India) was used for sub-culturing of bacterial strains. Onitrophenyl- -D-galactosides (ONPG) were purchased from Sigma-Aldrich USA for cell membrane assay. 2.2. Characterization of Nanoparticles Dry Titanium (IV) oxide-anatase nanopowder was procured from Sigma Aldrich, USA (CAS Number 637254). The suppliers data may be summarized as: 99.7% trace metal basis, particle size < 25 nm (BET), Surface area: 200-220 m2/g, density: 3.9 g/ml at 25 C, bulk density: 0.04-0.06 g/mL. The aqueous dispersion of the particles was subjected to TEM analysis. The morphological features and particle size of the procured nanoparticles were characterized by Scanning Electron Microscopy (FEI Sirion, Eindhoven, Netherlands). The surface area was measured using a Smart Sorb 93
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Single point BET surface area analyzer (Smart Instruments Co. Pvt. Ltd., Mumbai, India). 2.3. Preparation of Nanoparticle Dispersion Titanium dioxide nanoparticles procured were used as such to produce suspensions in deionized water (DI-water). The suspensions at four different concentrations at 10, 25, 50, and 100 ppm were made by means of ultrasonic vibration for 30 min using Crest ultrasonics system USA (132 kHz). 2.4. Growth Kinetics and Inhibition Studies The minimum inhibitory concentration (MIC), defined as the lowest concentration of material that inhibits the growth of an organism [12], was determined based on batch culture studies containing varying concentration of Titania nanoparticles in suspension (10, 25, 50, and 100 ppm). 50 ml Luria Bertani media in sterile side arm Erlenmeyer flasks (250 ml) were sonicated for 10 min after adding the nanoparticles to prevent aggregation of the nanoparticles. Subsequently, the flasks were inoculated with 1 ml of the freshly prepared bacterial suspension in order to maintain initial bacterial concentration 108 CFU ml1, and then incubated in an orbital shaker at 200 rpm and 30C. The high rotary shaking speed was selected to minimize aggregation and settlement of the nanoparticles over the incubation period. Lower rpm setting during incubation may also cause underestimation of the antimicrobial activity of the nanoparticles. Bacterial growth was measured as increase in absorbance at 600 nm determined using a spectrophotometer (CL-157 colorimeter, ELICO Company, Hyderabad, India). All these tests included a positive control (flask containing nanoparticles and nutrient media, devoid of bacterial inoculum) and a negative control (flask containing inoculum and nutrient media, devoid of nanoparticles). The negative controls indicated the microbial growth profile in the absence of nanoparticles. The absorbance values for positive controls were subtracted from the experimental values (flasks containing nutrient media, inoculum and nanoparticles) [13]. All the experiments were carried out in triplicates. 2.5. Protein Assay The extracellular protein was estimated by using Bradford assay method. Bacterial cells of E. coli, B. subtilis and P.aeruginosa were treated with 100ppm of titania nanoparticle solution for 14 h. Supernatant was collected after centrifugation (at 8000 rpm) for 15 min. For each sample, 200 l of the supernatant was mixed in 800 l of Bradford reagent. Optical density (at 595 nm) was measured after 10 min of incubation in the dark. BSA was used as a standard protein.

2.6. Integrity of Cell Membranes Bacterial cell membrane integrity was examined by determination of the release of cytoplasmic constituents of the cell. By monitoring change in absorbance at 260 nm, it is possible to estimate the amount of DNA and RNA released from the cytoplasm [14]. Bacterial cultures grown in nutrient broth were harvested by centrifugation at 11,000g for 10 min, washed and re-suspended in 0.5% NaCl solution. The final cell suspension was adjusted to an absorbance of 0.7 at 420 nm. The solutions of TiO2 nanoparticle and 0.5% acetic acid solution (control) were adjusted to pH 6.0 with 10% NaOH solution. A 1.5-ml portion of TiO2 nanoparticle solution and acetic acid solution was mixed with 1.5 ml of each bacterial cell suspension. The release of materials absorbing at 260 nm over time was monitored with a UV spectrophotometer (SANYO SP65 UV/VIS, Japan). 2.7. Inner Membrane Permeabilization Assays Inner membrane (IM) permeabilization was determined by measuring the release of cytoplasmic -galactosidase activity from microbes into the culture medium using ONPG as the substrate [15]. Bacteria grown to logarithmic phase in nutrient broth containing 2% lactose were harvested by centrifugation at 11,000g for 10 min, washed and resuspended in 0.5% NaCl solution. The final cell suspension was adjusted to an absorbance of 1.2 at 420 nm. The TiO2 NP dispersion and 0.5% acetic acid solution (control) were adjusted to pH 6.0 with 10% NaOH solution. TiO2 NP dispersions (1.6 ml) were each mixed with bacteria suspension (1.6 ml) and 30 mM ONPG solution (150l). The production of o-nitrophenol over time was determined by monitoring the increase in absorbance at 420 nm using SANYO SP65 UVVIS spectrophotometer (Japan). 2.8. Growth Medium and Sample Preparation for FTIR The microorganisms were grown in three different culture batches that contained 50 ml of Luria Bertani broth (Himedia Laboratories Ltd., Mumbai, India) with 100 ppm of Titania nanoparticles suspension in 250ml Erlenmeyer flasks at 37C in a shaker incubator overnight. Positive and negative control was also maintained through out the experiment. Bacterial cells were collected from the liquid cultures by centrifugation (Remi cooling centrifuge) at 10,000 rpm for 10 min. After removing the supernatants, the bacterial pellets were washed twice with phosphate buffer (pH 7; 1mM Na2HPO4/NaH2PO4 buffer). After second wash in phosphate buffer, samples for FTIR analysis were stored at -20C until lyophilized. Samples were then lyophilized using (Thermo Scienfic Co Micromodulyo freeze dryer, U.S.A). The samples were first pulverized into fine particles using mortar and pestle. The 1 mg of each sample was then mixed with 100mg KBr which extensively dried in microcentrifuge tubes using a Lyophilizer. These mixtures have been dried for an additional 2 h in the same microcentrifuge tubes. KBr based pellets were prepared by establishing pressure of 100 kg/cm2 (1200 psi) for about 5 min. Nicolet 6700 FT-IR Spectrometer (Thermo Scientific instruments groups, U.S.A) was used for analysis. Same protocol was followed for control without nanoparticle treatment. 3. RESULTS AND DISCUSSION 3.1. Characterization of Procured Nanoparticles The TEM analysis of Titania was shown in Fig. (1) showing an effective diameter of 20nm; uneven particles were noticed in 2 clumps. The surface area was determined to be 200-220 m /g. The high resolution SEM image of procured Titania nanoparticles is shown in Fig. (2). Nearly ellipsoidal to oval shaped nanoparticles was observed. The particles seemed to be in agglomerated condition. The difference between suppliers data and experimentally obtained data may be due to agglomeration of the particles while present in aqueous suspension.

Fig. (1). TEM analysis of Titania (Anatase) nanoparticle dispersion.

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Fig. (2). Scanning electron micrograph showing procured Titania nanoparticles.

3.2. Antimicrobial Activity of Titanium Dioxide Against E. coli, P.aeruginosa, and B. subtilis In further experiments, strains of Gram-negative bacteria E. coli, P. aeruginosa, and Gram positive bacteria such as B. subtilis were inoculated in liquid LB media supplemented with increasing dosage of titania nanoparticles. Increasing concentration of nanoparticles progressively retarded the growth of E. coli, P. aeruginosa, and B. subtilis (Figs. (3A), (3B), and (3C)). The concentration of 100 ppm was found to be inhibitory for bacteria among the three concentrations used, as it took about 4-6 h to initiate any noticeable growth. The steepness of the growth curve in the logarithmic phase, and the final cell concentration was also noticeably lower at 50ppm and 100ppm concentrations. 3.3. Prediction of Bacterial Growth in the Presence of Varying Concentrations of Titanium Dioxide Nanoparticles In the logarithmic phase the dynamic growth rate of a bacterial species is represented by the following equation ln N = ln N0 + t (1)

Where N is the bacterial cell count at time t, N0 is the initial cell count and is the growth rate constant for the bacteria. From the growth curve (Figs. (3A), (3B), and (3C )) of the bacteria at different concentrations of titania nanoparticles the logarithmic phase was identified between 6-14 h. The growth data in this time interval were plotted in the logarithmic scale (Figures not shown) to derive the values of dynamic growth rate () corresponding to various doses of the nanoparticles (x) studied. Linear relationships between x and (Figs. 4A, 4B, and 4C) was derived in the following form for E. coli, B. subtilis, and P. aeruginosa strains respectively: = ax + b = -0.0292x + 0.2281 = -0.0097x + 0.1666 = -0.0395x + 0.3241 (2)

Fig. (3A, 3B, and 3C). Effect of Titania nanoparticle concentrations on growth of E. coli, B. subtilis and P. aeruginosa.

it may be noted that for both the Gram negative strains a values were close, and for Gram positive B.subtilis it was one order lower compared to Gram negative strains. 3.4. Protein Assay of Nano Titania Interacted Bacterial Cells The extra cellular protein content from the uninteracted control cells of E. coli was measured to be 7.5 g/ml. The extracellular proteins extracted from fully grown culture in presence of 100ppm of titania nanoparticle concentrations, was found to be increased by 7.5g/ml for E. coli. The protein content of uninteracted control cells of P. aeruginosa was measured to be 18g/ml and nanoparticles treated cell was found to be increased by two fold to 44 g/ml. For B. subtilis the protein content of uninteracted cells was measured to be 14 g/ml and for the nanoparticle interacted cells it was 54g/ml.

The equations for the three strains respectively were noted as: (2A) (2B) (2C)

The value of a signifies dependence of growth rate on the concentration of the nanoparticles. Importantly all these cases negatively correlated with concentration of the particles. Increase in the nanoparticles concentration would significantly decrease the growth rate of all the three strains. Looking at the three a values,

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Fig. (4A, 4B, and 4C). Dynamic cell growth rate () of E.coli, B. subtilis and P. aeruginosa as a function of titania nanoparticle concentrations.

The result clearly showed that nanoparticle treated test solution had higher protein content than the untreated one in all the three cases. The increase in the protein content indicated that apart from the extracellular protein a certain amount of intracellular protein was also present in the nanoparticle treated test solution. The presence of intracellular protein in the test solution might be because of the action of nanoparticle on the integrity of microbial cell membrane. So further tests were carried out to study the integrity of the cell membranes in the treated cells. 3.5. Integrity of Bacterial Cell Membranes The cytoplasmic cell membrane is a structural component, which may be damaged and functionally invalid when bacterial suspensions are exposed to antimicrobial agents. If bacterial mem3 brane is compromised, ions such as K+ and PO4 tend to leach out first, and followed by large molecules such as DNA, RNA and

other materials. The release of these intracellular components with strong UV absorption at 260nm is an indication of membrane damage [14]. In our study we found that treatment of E. coli, P. aeruginosa and B. subtilis with 100ppm of titania nanoparticles resulted in a gradual release of intracellular components (Fig. 5). In detail, absorbance increased after 50 min of interaction at 260nm with 100ppm titania nanoparticle dispersion mixed with all three bacterial species. The DNA, RNA amount increased with time in the extracellular fluid. This may be due to the action of nanoparticle on the integrity on microbial cells. Few subtle differences were observed in the Gram positive and Gram negative organisms, due to the differences in cell wall structure and composition. The cell wall of E. coli and P. aeruginosa is made up of a thin membrane of peptidoglycan and an outer membrane composed of lipopolysaccharide, lipoprotein and phospholipids. But the cell wall of B. subtilis is composed of thick petidoglycan layer with teichoic acid, so for-

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galactosidase caused by titania was concentration-dependent (after about 50 min), which was agreeable with the assay of integrity of bacterial cell membranes and OM permeabilization of E. coli. Other workers [21] have reported similar results using chitosan. Results indicated that titania rapidly increased the permeability of the IM. TiO2 photocatalyst are known to damage cell membrane by lipid peroxidation resulting in a change of membrane permeability and fluidity that make the cell less able to absorb nutrient and more vulnerable to osmotic stress [6, 22]. 3.7. FTIR Study FTIR spectra of untreated and 100 ppm nanoparticles treated E. coli cells (Fig. (7A)) revealed the following bands: 2357.53 cm-1 shown in the nanoparticle interacted cells, which correspond to CH2 symmetric stretch mainly from lipids, with the little contribution from proteins, carbohydrates, nucleic acids where as it was absent -1 in the control; 1641.25.cm , which corresponds to Amide I (protein C=O stretching) absent in the control cells but present in the

Fig. (5). Release of cellular materials that absorb at 260 nm from E. coli, B. subtilis, and P. aeruginosa Suspensions treated with 100 ppm Titania nanoparticles dispersion.

Fig. (6). Release of cytoplasmic -galactosidase activity (measured by absorbance at 420 nm) of E. coli, B. subtilis, and P. aeruginosa cells trated with acetate100 ppm Titania nanoparticle solution.

eign molecules could easily enter the cell with making pores in the cell wall. So the rate of release of materials at 260 nm was the highest in case of B. subtilis. Our results evidenced that titania anatase nanoparticles could able to change the integrity of bacterial cell membranes and induce the release of intracellular component. Lu et al. [16] observed severe decomposition of cell wall and leakage of intracellular materials for cells exposed to UV-irradiated TiO2. Aldehydes produced by the peroxidation reaction could further damage proteins and other cell components. ROS are also generated and reacted with biomolecules, inactivate enzymes and damage DNA by direct reaction with the nucleic acids [17-20]. 3.6. Inner Membrane Permeabilization of Titania Nanoparticles When the IM is compromised, -galactosidase, an endoenzyme, could permeate the cytoplasmic membrane. The ability of titania nanoparticles to permeate the bacterial IM was evaluated through the production of o-nitrophenol (ONP) as a function of cytoplasmic -galactosidase release, with bacteria grown in lactose containing medium. When the bacterial cells were treated with titania nanoparticles (Fig. 6), a considerable release of the enzyme into the medium within 40 min was noted. There was immediate release of cytoplasmic -galactosidase followed by a progressive release for up to 60 min to reach a steady state, and then at a decreasing rate up to 100 min. In control suspensions, the release of -galactosidase increased at a decreasing rate up to 120 min after a lag of about 40 min. As shown in Fig. (6), the release of cytoplasmic -

nanoparticle treated cells; 1044.59 cm which corresponds to PO2 symmetric stretching: nucleic acids and phospholipids CO stretch: glycogen is present in control cells but absent in the nanoparticle treated cells. The bands at 970.85, 874.30, 743.24, 704.69, 647.06,and 546.75 cm-1 correspond to CN+C stretch: nucleic acids, N-type sugar; coupled furanosephosphodiester chain, Dipicolinic acid (DPA), NH2 & N-H wagging, and glycogen were present in the uninteracted cells whereas all of these bands were absent in the nanoparticle treated cells. All these structural changes may be attributed to the deformation of amino acids and cell membrane damage by 100 ppm titania nanoparticles dispersion. Fig. (7B) shows the FTIR band pattern of B. subtilis in which -1 also the 1404.67, 1234.19 cm bands which corresponds to COOsymmetric stretch: aminoacid side chains, fatty acids and PO2 asymmetric stretching: mainly nucleic acids with the little contribution from phospholipids were absent in the nanoparticles treated cells. Fig. (7C) shows the FTIR band pattern of P. aeruginosa, 542.89, 585.86, 663.64, 741.58, 918.83, and 1701.55 which corresponds to spectral assignments like S-S disulfide, Glycogen, CisRCH=CHR, Dipicolinic acid (DPA), Xylo-oligosaccharides; disubstituted Xylose residues and Ester C=O stretch: lipid, triglycerides were absent in the nanoparticle treated cells. The bands for OH stretching, amide II, DPA, Amide -I, and amino acids bands were conspicuously absent in the spectra.

-1

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Fig. (7A, 7B, and 7C). FT-IR analysis of E. coli, B. subtilis and P. aeruginosa before and after interaction with 100 ppm titania nanoparticle dispersion.

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CONCLUSION The present study reveals the batch growth kinetic data of titanium dioxide nanoparticles have promising effect on the growth profile of E. coli, B. subtilis and P. aeruginosa since these strains are abundant in nature and easily culturable. The dynamic growth rate of the bacteria was strongly dependent on the concentration of the nanoparticles dispersion, and the concentration dependence varied between the microbial strains. The inner membrane permeabilization assay, cell integrity assay and FTIR spectra suggested that titania caused the antibacterial effects through membrane damage mechanisms. The antibacterial effects of the nanoparticles essentially differed between the microbial strains due to structural differences of the outer membrane. The structure and surface properties of titania could be a cause for the physical disruptio n of cell membrane and interfere with membrane energy transduction resulting in microbial death. Further toxicity issues of titania nanoparticles to bacteria have to be addressed with a close insight on particle size and surface characteristics. REFERENCES
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Received: August 22, 2009

Revised: February 12, 2010

Accepted: March 10, 2010