J Photochem. Photobiol. B: Biol., 14 (1992) 369-379

369

Mode of photocatalytic bactericidal action of powderedsemiconductor Ti02 on mutans streptococci

T. Saito, T. Iwase, J. Horie and T. Morioka

Department of Preventive Dentistry, Faculty of Dentistry, Kyushu University 61, Fukuoka 812(Japan)

(Received October 18, 1991 ; accepted March 5, 1992)

Abstract

Powdered semiconductor TiO2 has a photocatalytic bactericidal capacity on some kinds ofbacteria, but its mechanism still remains unclear . The mode of its photocatalytic bactericidalaction on the mutans group of streptococci was investigated. Powdered TiO 2 had a bactericidalcapacity on all serotypes of mutans streptococci . Streptococcus sobrinus AHT was mainlyused for these experiments. The most effective concentration of TiO 2 was about 1 mgml- ' and, at this concentration, 10 5 colony-forming units of S. sobrinus AHT per millilitrewere completely killed within 1 min . In order to search for the mechanism of this effect,a high bacterial cell density (10' colony-forming units ml - ') was used in the followingstudies . "Rapid" leakage of potassium ions from the bacteria occurred parallel to thedecrease in cell viability . Protein and RNA were "slowly" released from bacterial cells fora reaction time up to 120 min . The pH of the reaction mixture decreased continuouslyto 4.5 after 120 min. Co-aggregation of S. sobrinus AHT and powdered Ti0 2 occurred athigh bacterial densities (above 108 colony-forming units ml -') . Aggregates gradually de-composed with light irradiation. Transmission electron microscopy of S. sobrinus AHT afterphotocatalytic action for 60-120 min indicated complete destruction of bacterial cells . Fromthese results, bacterial death appears to be caused by a significant disorder in cell membranesand finally the cell walls were decomposed .

Keywords: Powdered semiconductor TiO 2, photocatalysis, bactericidal action, mutansstreptococci, membrane permeability, potassium, co-aggregation, transmission electronmicroscopy.

1. Introduction

Solar energy conversion has recently attracted special interest, and there are manystudies of the photocatalysis induced by powdered semiconductors. Among thesephotocatalysts, powdered TiO2 possesses a strong oxidizing power for almost all kindsof organic substances [1-3]. Moreover powdered TiO2 has been reported to be capableof killing Saccharomyces cerevisiae, Escherichia coli and Lactobacillus acidophilus afterirradiation for 60-120 min using xenon or metal halide lamps, whose spectra are similarto sunlight [4] . As TiO2 is stable and harmless when swallowed by man, it is a possiblesubstance for application to the oral region . We have reported its bactericidal effecton the oral bacteria Streptococcus sobrinus AHT [5, 6], but until now there have been

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370

few studies on the mechanism of this bactericidal action of TiO 2 . The present studieswere performed in order to search for the possible mechanism of the bactericidalaction that TiO2 photocatalysis has on mutans streptococci .

2. Materials and methods

2.1. Organisms and growth mediaThe following strains of mutans streptococci were used : Streptococcus mutans

(serotypes c, a and f; GS5, LM7, OMZ175), Streptococcus rattus (serotype b; FA-1),S. sobrinus (serotypes d and g ; K1R, AHT) and Streptococcus cricetus (serotype a ;HS6). Of these strains, S. sobrinus AHT was mainly used for the experiments . Thestrains of mutans streptococci were grown overnight in brain heart infusion broth(BBL, Cockeysville, USA) which contained 8 mM K' . Aliquots of the overnightcultures were inoculated into fresh medium and incubated at 37 °C anaerobically untila stationary phase of growth was reached (approximately 1 X 109 colony-forming units(CFUs) ml -1 ) . The growth was monitored by measuring optical density . The cultureswere washed twice with saline (154 mM NaCl in double-distilled water) by centrifugationat 6000g for 5 min at room temperature, and the pellets were resuspended in saline .The cell suspensions were adjusted in glass vials (3 cm in diameter and 5 cm long)to the required cell density (105-10 10 CFUs ml -1) .

2.2. Photocatalytic reactionPowdered semiconductor Ti02 (mean primary particle size, 21 nm ; 70% anatase

type, 30% rutile type; pI 6.6; P25, Nippon Aerosil, Japan) was added to the salinementioned above and homogenized by sonication. The suspension of TiO2 was addedto the glass vials containing the cell suspension and adjusted to the required TiO 2concentration (0 .01-10 mg ml -1). The photocatalytic reaction was started by irradiatingthe vials with near-UV light including visible blue light (wavelength, 300-400 nm ; peak352 nm) and stopped by turning off the light . The light sources used were fluorescentlamps (20 W; fluorescent chemical lamp FL20S-BL, Toshiba, Japan) placed on bothsides of the vials at a distance of 3.5 cm to obtain a large amount of uniform lightenergy. The reaction mixture was stirred with a magnetic stirrer to prevent settlingof the TiO2 . All the apparatus were set in an incubator under aerobic condition andthe temperature of the reaction mixture was regulated at 37 °C . A bacterial suspensionwithout TiO 2 was irradiated as a control. After the light irradiation an aliquot of thereaction mixture was immediately diluted with saline (154 mM NaCl) and plated onbrain heart infusion agar plates (8 mM K'). In some experiments, KC1 (up to 200mM) and glutathione (GSH, 1 mM) were added to the saline and the brain heartinfusion agar plates. These plates were incubated anaerobically at 37 °C and thecolonies were counted after 2 days . The rest of the reaction mixture was filtered(Minisart; 0.2 µm pore size; Sartorius, Germany) and used for the following analyses.

2.3. Assays of the filtrate of the reaction mixtureThe potassium concentration in the filtrate of the reaction mixture was determined

by flame photometry (180-60 polarized zeeman atomic absorption spectrophotometer,Hitachi, Japan). 100% K' leakage was determined by sonication of the cells . Theprotein concentration in the filtrate was measured by the method of Lowry et al. [7]with bovine serum albumin (Fraction V, Sigma, USA) as a standard . The pH of thefiltrate was measured with a pH meter (F-15, Horiba, Japan) .

37 1

2.4. Determination of RNA in the reaction mixtureThe filtrate of the reaction mixture was mixed with perchloric acid (the final

concentration was 0 .5 N) and heated for 15 min at 90 °C with occasional stirring .The quantities of RNA in it were determined by reaction with orcinol [8, 9] usingRNA from bakers' yeast (type XI, Sigma, USA) as a standard . Aliquots of the reactionmixture were centrifuged and the quantities of RNA in the pellets (per 1 ml of reactionmixture) were also determined by the same method .

2.5. Assays for co-aggregation of bacterial cells with Ti02Mixed suspensions of S . sobrinus AHT cells and Ti02 were prepared as described

above. The final cell densities were adjusted to 101, 106 , 10', 10 8, 10 9 and 1010 CFUsml-'. The final TiO2 concentrations were adjusted to 0 .01, 0 .10, 1 .00 and 10.00 mgml-' . Co-aggregation in the reaction mixture was assessed with the naked eye soonafter mixing . After light irradiation, co-aggregation in the reaction mixture (S. sobrinusAHT, 109 CFUs ml-1 ; TiO2 , I mg ml-1) was analysed with a Coulter multisizcr(Coulter, USA) using a 30 Am aperture tube . The size distribution of 5000 co-aggregates(limited to 0.6-18 µm) was monitored at each irradiation time .

2.6. Transmission electron microscopySamples of the reaction mixture, which consisted of TiO 2 (1 mg ml- ') and S .

sobrinus AHT (109 CFUs ml- ') were centrifuged and pre-fixed in 2.5% glutaraldehydefor 2 h, washed three times with 0.1 M phosphate buffer (pH 7 .2) and post-fixed for2 h in 1% osmium tetroxide . The specimens were dehydrated in a graded series ofethanol and embedded in Epon 812. Ultrathin sections (60 nm) were made with anultramicrotome (Ultracut N, Reichert-Nissei, Austria) using a diamond knife, stainedwith uranyl acetate and lead citrate and studied in a transmission electron microscope(H-7000, Hitachi, Japan) at 100 kV accelerating voltage .

3 . Results

The most effective concentration of TiO 2 with respect to its photocatalytic bac-tericidal effect on S. sobrinus AHT (101 CFUs m-') was determined (Table 1) . Thephotocatalytic bactericidal action of TiO2 was strongly influenced by its concentration .

TABLE 1

Bactericidal effect of TiO 2 photocatalysis at various concentrations on Streptococcus sobrinusAHT (101 CFUs m1'')

Mean±SE (n=3) .

PowderedTiO2(mg MI-1 )

log(CFUs nil - ') for the followingirradiation times

0 min 3 min

0 4.8±0 .1 4.8±0 .10 .01 4.9±0 .1 4.3±0 .10.10 4.8±0 .1 2.1±0.51 .00 5.1±0 .1 <1

10.00 5.1±0 2.5±0.8

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Over 10 mg ml - ' and under 0.01 mg ml - ' the bactericidal effect obviously decreased .In the former case the reaction mixture formed an emulsion and strong light scatteringoccurred at the surface. Therefore the light could not reach the TiO 2 in the interiorof the reaction mixture . The killing effect occurred efficiently in translucent or transparentmixtures in which the concentration of Ti0 2 was 1 mg ml-1 . The most effectiveconcentration of TiO2 (1 mg ml- ') was the same in the experiments in which a highbacterial cell density (10' CFUs ml - ') was used; therefore this concentration was usedin the following studies .

The photocatalytic bactericidal effect of Ti0 2 (1 mg ml- ') at a low density of S .sobrinus AHT is presented in Fig . 1. Within 1 min S. sobrinus AHT (105 CFUs ml-')were completely killed by the photocatalytic action of TiO 2. On the contrary, therewas no decrease in the number of CFUs in the experiment in the presence of TiO 2without light irradiation and in the control with light irradiation alone . This bactericidaleffect was about the same in each strain of mutans streptococci used in this study,including all serotypes . Their survival rates after photocatalytic treatment for 3 minwere less than 0 .1% in the same experimental set-up as in Fig . 1 .

In order to search for a possible mechanism for this photocatalytic bactericidaleffect, the leakage of intracellular substances was examined . To detect a small amountof leaked substances a high bacterial cell density (10' CFUs ml - ') was used in thefollowing studies. Under this condition, bacterial cells and TiO 2 formed visible co-aggregation soon after they had been mixed (Table 2) . Then it took a much longertime for TiO2 to kill bacteria, as Ti02 particles in the interior of co-aggregates werenot irradiated (Fig . 2). In the control, there was only a slight decrease in the numberof CFUs after light irradiation for more than 10 min . At 0 min, the number of CFUsof the experiment was less than the number of CFUs of the control, caused by co-aggregation in the experiment .

In order to determine the change in cell membrane permeability, potassium leakagefrom S. sobrinus AHT cells was examined (Fig. 3) . Immediately after the light irradiationwith TiO2 , K' started to leak from the bacterial cells. After 5-10 min all K` hadleaked out. The number of CFUs, as depicted in Fig . 2, was converted into the ratioof the number of CFUs after each min to the number of CFUs at 0 min and representedin Fig. 3. The ratio is an estimation of cell viability, even though it is not totally

E a

C0 t0 0

2

3Irradiation time (min)

Fig . 1 . Bactericidal effect of TiO2 photocatalysis (1 mg ml-') on S. sobrinus AHT (10' CPUsml-') : 0, experiment containing powdered TiO2 with light irradiation; 0, control in the presenceof powdered Ti0 2 without light irradiation ; A, control without TiO2, light irradiation alone .

TABLE 2Co-aggregation of Streptococcus sobrinus AHT and powdered Ti0 2

-, no visible co-aggregate; small co-aggregates had an approximate size of less than 1 mm; largeco-aggregates had an approximate size of greater than 1 mm .

n 9di

6E 7N

C 6Cc gE0 4C0 30O 2

373

-r, i03510

30

60

90

120Irradiation time (min)

Fig. 2. Bactericidal effect of TiO, photocatalysis (1 mg ml - ') on S. sobrinus AHT (10' CFUsml-1 ) : 0, experiment containing powdered TiO, with light irradiation ; A, control without TiO,,light irradiation alone . The bars indicate standard error (SE) (n=4) .

accurate because of co-aggregation . K' leakage paralleled the loss of cell viability .However, the loss of cell viability was also seen in reaction mixtures which containedhigh concentrations of K' (up to 200 mM) . Moreover, when bacterial cells were putinto the medium containing high K' (up to 200 mM) immediately after photocatalyticreaction for 1 min, they could no longer grow (data not shown) as in a mediumcontaining low K' . Glutathione, which has been known to be an essential cofactorfor "K' channels" in E . coli [10, 11], was also without effect when supplementedalone or together with K' immediately after photocatalytic reaction for 1 min .

Protein release from S. sobrinus AHT, induced by TiO, photocatalysis, was gradualup to about 100 µg ml - ' in the filtrate of the reaction mixture after reaction for 120min (Fig. 4). In the case of light irradiation alone, no protein release was recognized .

RNA was released from S. sobrinus AHT up to about a concentration of 12 µgml- ' in the filtrate of the reaction mixture after reaction for 120 min (Fig . 5). In thecase of light irradiation alone, no RNA release was recognized . The RNA quantity

PowderedTiO2(mg ml - ')

Co-aggregates for the following S . sobrinus AHT concentrations

0 CFUs 10' CFUs 108 CPUs 10' CPUs 108 CFUs 10' CPUs 10 10 CPUsml- ' ml-1 ml - ' ml- ' mi-1 ml - ' ml''

0.01 - Small -0.10 - Small Large Large Small1.00 - Large Large

10.00 - - - Large

374

9 0.25E0 0.20Ce

013 ~=

u0.10

s0 UO

COAS

Y

m0

d-- 100

0 3 5

10

30Irradiation time (min)

Fig. 3. Potassium leakage and loss of ce11 viability induced by TiO 2 photocatalysis, where ratioof CFU means the ratio of the number of CFUs at each minute to the number of CPUs at 0min: •, K' in the filtrate of the experiment with TiO2 (1 mg nil - ') with light; A, K* in thefiltrate of the control (light irradiation alone); 0, ratio of the number of CFUs at each minuteto the number of CFUs at 0 min in the experiment with TiO 2 (1 mg ml - ') with light ; Es, ratioof the number of CFUs at each minute to the number of CFUs at 0 min in the control (lightirradiation alone) . The bars indicate SE (n=4) .

EO)3 40

z

so

20

30

60

90

120

0 10 30

60

120Irradiation time (min)

Irradiation time (min)

Fig. 4. Protein release from S sobrinus AHT (109 CFUs ml-') induced by TiO2 photocatalysis :, experiment containing TiO2 (1 mg nil - ') with light irradiation ; A, control without TiOb light

irradiation alone .

Fig. 5. RNA in the filtrate and the pellet of the reaction mixture : •, RNA in the filtrate ofthe experiment with Ti02 (1 mg nil- ') with light ; A, RNA in the filtrate of the control (lightirradiation alone) ; 0, RNA in the pellet from 1 ml of the experimental mixture ; A, RNA inthe pellet from 1 nil of the control mixture .

in the pellets (per 1 ml of reaction mixture) is also presented in the same figure .There was a 50% reduction in RNA in the pellet after reaction for 60 min, and after120 min the reduction was about 85% . In the control, with light irradiation alone,there was no decrease in RNA in the pellets. The quantity of RNA that appeared

in the filtrate did not match that which disappeared from the pellet. When a solutionof RNA from bakers' yeast was phototreated with Ti0 2 under the same circumstancesfor 60 min, 50% of it could not be detected by the orcinol reaction . Apparently, inthe experiments with the bacteria, RNA also decomposed .

The pH change of the reaction mixture is shown in Fig . 6 . The pH of the reactionmixture decreased from 6 .8 (0 min) to 4.5 (120 min). In the experiments using 10%glucose or 50% methyl alcohol solution in water, which are known to be decomposedinto CO2 and H 2 by Ti0 2 [2, 3], the pH decreased from 6.5 to less than 4 in thesame experimental set-up without bacteria (data not shown) . The pH decrease in Fig .6 was probably due to the production of CO2 via carboxylic acid [2, 3] from constituentsof bacterial cells . In the experiment containing TiO2 but not bacteria and in the controlcontaining bacteria but not TiO,, there was no pH change during light irradiation for120 min .

In the cases of higher bacterial cell densities (above 10s CFUs ml - '), significantco-aggregation was observed with the naked eye soon after mixing . Table 2 shows theconditions required to cause co-aggregation of S. sobrinus AHT and TiO2. After lightirradiation the visible co-aggregates were decomposed . Figure 7 shows one case ofsuch decomposition. The most frequent size and the maximal size are presented . Thesize of the co-aggregates decreased at a high rate during the first 10 min of treatment,then remained constant for the next 20 min and thereafter decreased again but at amuch lower rate. Finally the particle size became the same as that in the case of theTiO2 suspension or the bacterial suspension which formed small aggregates in saline .

Figure 8 represents transmission electron microscopy (TEM) findings for S .sobrinusAI-IT after the photocatalytic action of TiO 2. After treatment for 30 min, somemorphological changes were observed in a few bacterial cells . The chromosomal regiongathered at the center of the protoplasm, surrounded by an area containing a net-like structure . The protoplasm had some electron-translucent portions . No disrupted

7.0-

6 .0

pH

5 .0

4 .

0 10 30

60

90Irradiation time (min)

120

375

0 10 30

60

120

Irradiation time (min)Fig. 6, The pH changes of the reaction mixture induced by Ti0 2 photocatalysis: •, experimentcontaining S.sobrinus AHT (10° CFUs ml - ') and powdered T10 2 (1 mg nil -') with light irradiation ;A, control without TiO2; •, powdered TiO2 (1 mg ml -') without bacteria .

Fig. 7. Size of co-aggregates consisting of S. sobrinus AHT (10° CFUs ml-') and powdered MO,(1 mg ml -') after light irradiation. The most frequent size of co-aggregates ranging from 0 .6to 18 µm was determined after counting 5000 co-aggregates (0). The case of bacterial suspension(A), and the case of Ti0 2 suspension (U) are represented in the left-hand corner ; these werenot irradiated . The bars indicate the maximal size of a co-aggregate after counting 5000 co-aggregates.

376

(a) (b)

(c)

(d)Fig. 8. TEM findings of S. sobrinus AHT with powdered T102 after light irradiation. Theirradiation time is indicated in the right-hand corner . Fine particles of TiO2 , labelled T, wereexhibited. A few cells, labelled B, were observed to have bad their cell wall structure partiallybroken after 60 min . Many cells were disrupted after 120 min .

377

cell wall was recognized . After 60 min, the majority of cells exhibited the samemorphological change but to a greater extent . A few cells had their cell wall structurepartially disrupted . After 120 min, no chromosomal complex was observed in themajority of cells, the central portion was hollow, the protoplasm revealed a coarseappearance, and many cells were evidently disrupted . In the control without Ti02 ,the protoplasm had small electron-translucent portions after light irradiation for 30min. These portions increased after longer irradiation . No disrupted cell wall wasrecognized until after treatment for 120 min .

4. Discussion

The wavelength range of the light used in this study was from 300 to 400 rim(peak, 352 nm) . Such near-UV light, free of far-UV light under 300 nm which isabsorbed by nucleic acids, does not cause severe damage to organisms . Generally,semiconductors can be excited by light of a shorter wavelength than that correspondingto the band gap energy of each semiconductor [4] . The band gap energy of Ti0 2 is3.0 eV, which is equivalent to light of 415 nm wavelength . Hence the fluorescentchemical lamp used in this study should be very efficient for the photocatalytic reactionof TiO2 in spite of its low output of 20 W [12] .

The powdered Ti02 used in this study (P25) consists of ultrafine particles (21nm, i .e . about one twentieth of the bacterial size ; see Fig . 8). Therefore it has moresurface area for the photocatalytic reaction and has a more efficient bactericidal effectthan larger Ti0 2 particles [12].

TiO2 photoreacts with almost all organic substances in aqueous solutions [2, 3] .Phosphate ions suppress the adsorption of Ti0 2 onto basic a-amino acid [13] . In fact,when phosphate or broth was added to the reaction mixture, the bactericidal effectwas much decreased (data not shown) . Therefore a simple saline solution was usedas the medium in this study .

Co-aggregation, as observed in this study, might have a similar mechanism to thatalready reported on powdered hydroxyapatite and oral streptococci [14, 15] . Powdereddental enamel, powdered hydroxyapatite and powdered T102 are all amphotericelectrolytes and their isoelectric points are about the same (pH 6.8 [16], pH 7.4 [17]and pH 6.6 respectively) . In addition, as about 7% of the cell wall of S . mutans consistsof basic a-amino acid such as lysine, arginine and histidine [18], they could very wellbe the adsorptive sites of TiO 2 [13] . The adsorption of TiO 2 onto bacterial cells maybe necessary for its bactericidal action because the photocatalytic reaction is inducedon the surface of TiO2. In view of the large species differences in the adsorption ofbacteria to hydroxyapatite [17, 19], the affinity of bacterial cells to TiO2 probablyinfluences the bactericidal effect of TiO 2 . There is a report on the differences of thephotocatalytic bactericidal effects of Ti0 2 on other strains of mutans streptococci [20] .In this study, there were no differences in the bactericidal effects between the strainsof mutans streptococci covering all serotypes. This was probably because killing tookplace too quickly under our experimental conditions.

The accumulation of K' is a universal characteristic of living systems [21-25]. Inbacteria, many previous studies have demonstrated that the loss of K' is followed bythe loss of cell viability [10, 11, 23-25] . Therefore K* leakage was examined in thisstudy as an indicator for membrane permeability changes . It occurred in a short timeparallel to the loss of cell viability. However, the addition of K* and/or glutathione[10, 11] could not maintain cell viability and thus K* leakage per se cannot be a single

378

cause of bacterial death. Moreover, as leakage of intracellular protein and RNA alsotook place without a lag time and progressed gradually, it is unlikely that this permeabilitychange is specific for K'. Other disorders occurring after or with the K' loss alsoseems to cause cell death . We shall interpret these results as follows . Around cellmembranes of the bacteria, which form the permeability barrier, there is a cell wallconsisting of peptidoglycan layers . As the primary particle size of TiO2 used in thisstudy is 21 too, TiO2 cannot be attached to the cell membrane directly. Consideringthe TEM findings and the size of TiO2, it takes more than 30 min for TiO 2 to reachthe membrane after decomposition of the peptidoglycan layer . However, the cell deathoccurred within 1 min. Therefore it is reasonable to assume that the membrane isdamaged by reactive oxygen species such as superoxide (0 ; -) or perhydroxyl radicals(HOZ) which are known to be produced from water around TiO 2 photocatalysis [26,27] and that this membrane oxidation causes gross disruption of the main permeabilitybarrier. The slow leakage of RNA and protein, compared with that of K', was probablycaused by their physical macromolecular nature .

As is evident from the electron micrographs, cell walls of S. sobrinus AHT weredestroyed after photocatalytic treatment for 60-120 min . This phenomenon is consistentwith the decomposition of co-aggregates presented in Fig . 7 . Considering all the resultsin this study, the cell wall destruction is not essentially concerned with the loss ofcell viability but is a secondary phenomenon .

Powdered TiO2 is not costly and it has been widely used in paints and cosmeticsas a white pigment or an absorbent of UV light. The safety of powdered TiO2 forman has been recognized for many years [28-30] . It might find some use in the oralregion, especially for the purpose of inactivation of dental plaque . As TiO 2 is activatedby visible blue light or sunlight, it has possibilities for application in many fields .

Acknowledgments

We express sincere appreciation to the late Dr . Y. Nara (Associate Professor,Department of Preventive Dentistry, Kyushu University) for his helpful advice andsuggestions. We also thank Dr. R. Kimura (First Department of Conservative Dentistry,Kyushu University) for his help in the TEM study . This study was supported in partby a Grant (01771855) from the Ministry of Education, Science and Culture in Japan.

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