Catalysts 2013, 3, 310-323; doi:10.3390/catal3010310
OPEN ACCESS

catalysts
ISSN 2073-4344
www.mdpi.com/journal/catalysts
ArticleBroad Spectrum Microbicidal Activity of Photocatalysis by TiO2
Ryuichi Nakano 1,2,†, Masayuki Hara 2,†,*, Hitoshi Ishiguro 2,3, Yanyan Yao 2,3, Tsuyoshi Ochiai 2,4,
Kazuya Nakata 2,4, Taketoshi Murakami 2, Jitsuo Kajioka 2, Kayano Sunada 2,5,Kazuhito Hashimoto 5,6, Akira Fujishima 2,4 and Yoshinobu Kubota 2,3
1

2

3

4

5

6

Department of Microbiology and Immunology, School of Medicine, Teikyo University, 2-11-1 Kaga,
Itabashi-ku, Tokyo 173-8605, Japan; E-Mail: nakano@med.teikyo-u.ac.jp
Kanagawa Academy of Science and Technology, 3-2-1 Sakado, Takatsu-ku, Kawasaki,
Kanagawa 213-0012, Japan; E-Mails: h1496@med.yokohama-cu.ac.jp (H.I.);
yanyan01@med.yokohama-cu.ac.jp (Y.Y.); pg-ochiai@newkast.or.jp (T.O.);
pg-nakata@newkast.or.jp (K.N.); pg-murakami@newkast.or.jp (T.M.);
pg-kajioka@newkast.or.jp (J.K.); kayano@light.t.u-tokyo.ac.jp (K.S.);fujishima@newkast.or.jp (A.F.); kubotayo@med.yokohama-cu.ac.jp (Y.K.)
Department of Urology, Graduate School of Medicine, Yokohama City University, 3-9, Fukuura,
Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
Division of Photocatalyst for Energy and Environment, Research Institute for Science andTechnology, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba,
Meguro-ku, Tokyo 153-8904, Japan; E-Mail: hashimoto@light.t.u-tokyo.ac.jp
Department of Applied Chemistry, Faculty of Engineering, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8656, Japan† These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: pg-hara@newkast.or.jp;
Tel.: +81-44-819-2041; Fax: +81-44-819-2070.
Received: 3 December 2012; in revised form: 25 January 2013 / Accepted: 6 March 2013 /Published: 21 March 2013

Abstract: Photocatalytically active titanium dioxide (TiO2) is widely used as a self-cleaningand self-disinfecting material in many applications to keep environments biologically clean.
Several studies on the inactivation of bacteria and viruses by photocatalytic reactions havealso been reported; however, only few studies evaluated the spectrum of the microbicidal
activity with photocatalysis for various species. There is a need to confirm the expectedCatalysts 2013, 3

311

effectiveness of disinfection by photocatalysis against multidrug-resistant bacteria and viruses.
In this study, microbicidal activity of photocatalysis was evaluated by comparing the inactivationof various species of bacteria and viruses when their suspensions were dropped on the surfaceof TiO2-coated glass. Gram-positive bacteria, e.g., methicillin-resistant Staphylococcus aureus,
vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae,
were easily inactivated by photocatalysis, whereas some gram-negative bacteria, e.g.,
Escherichia coli and multidrug-resistant Pseudomonas aeruginosa, were gradually inactivatedby photocatalysis. Influenza virus, an enveloped virus, was significantly inactivated byphotocatalysis compared with feline calicivirus, a non-enveloped virus. The effectivenessof microbicidal activity by photocatalysis may depend on the surface structure. However,they are effectively inactivated by photocatalysis on the surface of TiO2-coated glass. Ourdata emphasize that effective cleaning and disinfection by photocatalysis in nosocomial
settings prevents pathogen transmission.
Keywords: photocatalysis; disinfection; drug resistant bacterium; virus; UV-A; TiO2

1. Introduction
Infectious diseases caused by antimicrobial resistance bacteria are becoming one of the major
problems during use of antibiotics worldwide [1,2]. Survival of these microbes on environmental
surfaces and medical devices leads to nosocomial disease transmission and is thought to contribute to the
90,000 annual deaths from nosocomial pathogens [3–5]. Nosocomial infections are frequently associated withresistant organisms, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistantEnterococcus spp. (VRE), penicillin-resistant Streptococcus pneumoniae (PRSP), Serratia marcescens,multidrug-resistant Acinetobacter baumannii (MDRA), and multidrug-resistant Pseudomonas
aeruginosa (MDRP) [6,7]. MRSA and PRSA are also a prevalent cause of community-acquired infection [8].
Currently, new resistance mechanisms, e.g., the New Delhi metallo-β-lactamase-1 (NDM-1), haveemerged among several gram-negative bacteria and spread worldwide [9]. Viable pathogens of theseorganisms in the environment have a significant role in nosocomial transmission, especially for
immunocompromised patients.
Furthermore, viruses such as the influenza virus (IFV) and norovirus (NoV) spread across theworld [10,11]. These viral species can be transmitted to susceptible hosts via contaminated inanimate
surfaces. Control of viral contamination is important to prevent nosocomial transmission. Respiratory
and enteric viruses have been reported to survive for days on surfaces, and viral infections are due totransfer from these surfaces to susceptible persons [12–14]. IFV and NoV can be transmitted by direct
and indirect contact with contaminated surfaces and/or aerosol and is linked to the outbreaks in
hospitals and long-term care facilities [15,16].
These infectious diseases are a threat to human health, and indeed, outbreaks and serious clinicalcases have occurred [17]. Effective disinfection of healthcare equipment and surfaces is critical for
preventing transmission of potential pathogens [18]. Therefore, new antibacterial and antiviralmaterials or new countermeasures against such microbes are urgently required to disinfect the surface.

Catalysts 2013, 3

312

Photocatalytically active titanium dioxide (TiO2) is widely used as a self-cleaning andself-disinfecting material in many applications to keep environments biologically clean [19–21].
Photocatalysis mainly uses a semiconductor such as TiO2 which can absorb UV light (k < 400 nm) andcan photo-stimulate redox reactions on its surface producing Reactive Oxygen Species (ROS) such ashydroxyl radical (·OH), superoxide radical (·O2−) [22] and singlet oxygen (1O2) [23], and the
production of them may contribute to the biocidal activity with strong oxidative activity and destroyorganic compounds [24].
This photocatalytic oxidizing power has been applied for the removal of several toxic substancesfrom water and air [25–27]. Many photocatalytic disinfection studies in which TiO2 was used against
bacteria and fungi have been reported [28,29]. Many products that utilize TiO2 photocatalysis havebeen commercialized in many countries. For example, TiO2-coated tiles have been used as
self-cleaning and antibacterial materials [30]. While we have reported the virucidal activity ofphotocatalysis for IFV [31] and bacteriophages [32,33], few studies have addressed viruses [34].
Recently non-TiO2 photocatalysts (i.e., ZnO and ZnS) reported as significant progress archived indevelopment were also observed their antimicrobial activity [35,36].
Thus, photocatalysis is expected to be applied by antimicrobial materials for many harmful
pathogens. To use the superb photocatalytic effect of TiO2 is a conceptually feasible technology forthis material, and its production at industrial scale is easy and inexpensive. However, there is no study
comparing the disinfecting activity of photocatalysis for bacteria and virus using typical TiO2 sample
in the same condition, and the effect on drug-resistant bacteria has also not been demonstrated. This
study was designed to elucidate the antimicrobial spectrum and the microbicidal activity ofphotocatalysis on different bacteria species which were bacteria (i.e., gram-positive cocciand gram-negative rods included drug-resistant bacteria) and viruses (i.e., enveloped andnon-enveloped viruses).
2. Results and Discussion
2.1. Bactericidal Activity of Photocatalysis by TiO2-Coated Glass
We investigated the bactericidal activity of photocatalysis against different bacteria species thatcause an infectious disease at clinical sites. The tested antimicrobial-resistant bacteria species cause
opportunistic infections by contact infection and it is very important to cleanse them in a clinicalenvironment. Tested bacteria species on control glass were exposed to 0.1 mW/cm2 UVA; however,significant inactivation was not observed, it means the strains were stable under the condition(Figure 1). Photocatalytic inactivation of different bacteria species was evaluated with TiO2-coatedglass under UVA light intensity of 0.1 mW/cm2. All bacteria species could be inactivated by treatment
with TiO2 under UV irradiation in a time-dependent manner. We found that TiO2 exhibited asignificantly better performance to reduce the number of surviving bacteria when compared withunirradiated (0 h) (Figure 1, p < 0.05). Viable bacterial counts of gram-positive cocci (GPC) such as
MRSA, VRE, and PRSP were reduced; the log reduction value (LRV) of them was 1.4–2.8 logs after
2 h, respectively. The number of gram-negative rods (GNRs) such as Escherichia coli, S. marcescens,
MDRP, MDRA, and NDM-1 producing Klebsiella pneumoniae gradually decreased, the LRV was

Catalysts 2013, 3313

between 0.8 and 1.5 logs after 2 h. It is interesting to note that there was a significantly different
bactericidal activity between GPC and GNR. This finding suggests differences in the mode of
antimicrobial action.
Figure 1. Photocatalytic inactivation of different bacteria with (a) control glass or(b) TiO2-coated glass under black-light illumination (UVA light intensity of 0.1 mW/cm2).
The data shown in the figure are the average values of three experiments. Error bars:
standard deviations of three replicate experiments. * p < 0.001, 2 h, 4 h, 8 h vs. 0 h exposed
to 0.1 mW/cm2 UVA.
(a) Control glass0h

Bacterial survival (log CFU/mL)

1.E+062h4h8h

1.E+05
1.E+04
1.E+03
1.E+02
1.E+01

Detection limit

1.E+00
Ecoli

Serratia MDRP MDRA NDM1(b) TiO2-coated glass

Bacterial survival (log CFU/mL)

1.E+06

*
*
*

0h
*
*
*

*
*
*

2h

4h
*
*
*

MRSA

8h
*
*
*

*
*
*

VRE

PRSP

*
*
*

*
*
*

1.E+05
1.E+04
1.E+03
1.E+02
1.E+01

Detection limit

1.E+00
Ecoli

Serratia MDRP MDRA NDM1

MRSA

VRE

PRSP

In particular, GPCs of MRSA, VRE, and PRSP were significantly decreased, i.e., number ofsurviving cells. They were inactivated below the detection limit (<1 log10 colony-forming units/mL)
within 4 h. Many MRSA infections occur with a higher incidence rate in hospitals, healthcare
facilities, or long-term care facilities. MRSA is transmitted by contact with a colonized carrier(someone carrying the infection on the skin but who is not infected), or a contaminated object, e.g.,clothing and environmental surfaces. The bactericidal activity of photocatalysis would inactivate
MRSA effectively at environmental surfaces in hospitals and could prevent the spread of nosocomial
infections. The effectiveness of the bactericidal activity by photocatalysis could be one of the infection
control strategies for preventing environment-associated infections in healthcare facilities. VRE andCatalysts 2013, 3

314

PRSP could also be inactivated by photocatalysis and it can be expected to prevent their spread
as MRSA.
Except for MDRA, the number of surviving cells of GNRs gradually decreased compared to that of
GPCs under UV irradiation with TiO2. The number of cells of MDRAs significantly decreased as thenumber of GPCs reached a value below the detection limit within 4 h. The number of cells of all tested
GNRs decreased to below the detection limit after 16 h (data not shown). The difference in thebactericidal activity for GPC and GNR could be associated with their surface layer structure(Figure 2 and Table 1). The structure of S. aureus, as a representative GPC, is constituted of thick
peptidoglycan layer, which covers more than 50%, usually almost 90%. The peptidoglycan layer in thebacterial cell wall has a crystal lattice structure formed from linear chains of two alternating amino
sugars, N-acetylglucosamine and N-acetylmuramic acid. The structure of E. coli, as a representative
GNR, consists of a thin peptidoglycan layer (which is much thinner than that in GPCs) and an outermembrane containing lipopolysaccharide (LPS) in its outer leaflet and phospholipids in the
inner leaflet.
Figure 2. Structure of gram-positive and gram-negative bacteria: (a) Gram-positive bacteria
have a thick wall composed of peptidoglycans; and (b) gram-negative bacteria have an outermembrane and a thin wall composed of peptidoglycans.

Table 1. Comparative characteristics of gram-positive and gram-negative bacteria.
Characteristic
Thickness of wall
Number of layers
Peptidoglycan content
Lipid and lipoprotein content
Lipopolysaccharide content

Gram-positive
thick (20–80 nm)
1
>50%
0%–3%
0

Gram-negative
thin (10 nm)
2
10%–20%58%
13%

The outer membrane of GNRs is unique due to the high content of lipids. The relative resistance ofthe cell wall of GNR to dissociation by detergents, disinfectants, and antiseptics is well
documented [37,38]. It is proposed that the presence of large amounts of LPS and proteins, together
with a very small amount of phospholipids in the outer leaflet of the outer membrane, is the decisive

Catalysts 2013, 3

315

factor in the effective detergent resistance of this membrane [37]. GNRs are generally more resistant todisinfectants than GPCs due to outer membrane barrier, but the resistant mechanism is not wellcharacterized. Although the microbicidal activity by photocatalysis has not been studied thoroughly andthe mechanism of the bactericidal process is still unknown [27,39], there are some suggestions andevidences for the steps leading to cell inactivation. TiO2 photocatalysis generates various ROS asfollows [24].
Reductive reactions:O2 + e− → O2−
O2− + H+ ֎ ·HO2
·HO2 + e− + H+ → H2O2
Oxidative reactions:OH− + h+  ·OH
These ROS can decompose organic compounds and extinguish cellular activity. Recent works
attributed that the TiO2 photocatalytic action induced significant damage in cell membranes by ROS,
followed by loss of essential functions such as respiratory activity and bacterial lysis leading to bacterial
death [24,25,40]. Sunada et al. [41] demonstrated the TiO2 photocatalytic inactivation of E. coli
endotoxin, which is a constituent of the outer membrane of GNRs. This finding suggests that the cellwall damage might take place prior to cytoplasmic membrane damage. After the outer membrane has
been disordered and partially decomposed (Figure 2), ROS gain access to the cytoplasmic membrane,
causing the cell to die. However, while the outer membrane protected GNRs, GPCs have a thick
peptidoglycan layer instead of an outer membrane; these ROS would presumably easily penetrate thecytoplasmic membrane and attack the cell membrane directly. The difference in their structure may beof decisive influence on the survival abilities of bacteria.
Some GNRs have flagella, which are long filamentous protein structures projected from thecytoplasmic membrane, constituted of microtubules and surrounded by the cell membrane (Figure 2).
Bacterial flagella provide mobility for capturing nutrients and avoiding harmful substances tosurvive [42]. In this study, MDRA is the exceptional strain among these tested GNR bacteria because
it has does not have flagella surrounding the cell membrane and it is inactivated by photocatalysis
within 4 h. It is suggested that the lack of flagella at the surface structure of MDRA may be part of thereason for higher bactericidal activity by photocatalysis. Alternatively, other structures such as capsule,pilus, and bacterial size and an antioxidative defense system [43] may be associated with theeffectiveness of bactericidal activity. Further study is necessary to clarify the mechanism of thephotocatalysis reaction to exercise a decisive influence on bactericidal activity by analyzing the effects
against the cell membrane surface.
2.2. Virucidal Activity of Photocatalysis by TiO2-Coated Glass
We investigated the virucidal activity of photocatalysis against IFV and feline calicivirus FCV(a surrogate for NoV) using purified and adjusted suspensions. Tested viruses on control glass werealso stable under the UVA light intensity of 0.1 mW/cm2 (Figure 3). As shown in Figure 3,
photocatalytic inactivation of these viruses was evaluated with TiO2-coated glass under UVA lightCatalysts 2013, 3

316

intensity of 0.1 mW/cm2. The viruses were inactivated by the treatment with TiO2 under UV irradiation
in a time-dependent manner, as shown for bacteria. The experiments were relatively reproducible andthis reduction was statistically significant when compared with unirradiated (0 h) (Figure 3, * p < 0.001).
The LRV of IFV at 4 h was 3.6 and the titer decreased to below the detection limit within 8 h, while
the LRV of FCV at 8 h was only 1.7. The FCV titer decreased to below the detection limit after 16 h. It
was concluded that the FCV titer was insignificantly reduced by the photocatalysis effect.
Figure 3. Photocatalytic inactivation of influenza virus (IFV) and feline calicivirus (FCV)with (a) control glass or (b) TiO2-coated glass under black-light illumination (UVA lightintensity of 0.1 mW/cm2). The data shown in the figure are the average values of threeexperiments. Error bars: standard deviations of three replicate experiments. * p < 0.001, 4 h,
8 h, 16 h vs. 0 h exposed to 0.1 mW/cm2 UVA.
(a) Control glass0h

1.E+08

4h

8h

16h

Virus titer (log PFU/mL)

1.E+07
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02Detection limit

1.E+01IFV(b) TiO2-coated glass

FCV0h

1.E+08
*

*

4h

8h

16h

*
*

*

*

Virus titer (log PFU/mL)

1.E+07
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02Detection limit

1.E+01IFV

FCVThese data demonstrate that IFV is faster inactivated by photocatalysis than FCV. The low virucidal
activity of photocatalysis against the non-enveloped virus was not surprising considering previous
studies on biocides. IFV, an enveloped virus, consists of a phospholipid bilayer membrane withglycoprotein spikes surrounding the capsid. FCV, a non-enveloped virus, has a capsid that consists of aprotein and lacks an envelope that surrounds it. Although enveloped viruses are effectively eliminatedby solvent/detergent treatment, non-enveloped viruses are not affected by this treatment [44].

Catalysts 2013, 3

317

Non-enveloped viruses, without a lipid coat, are resistant to most of the viral inactivation methods thathave been described to date [45,46]. These data indicate that the difference in the effectiveness toenveloped and non-enveloped viruses by photocatalysis is the same as that for disinfectants. It issuggested that the virucidal activity of photocatalysis may depend on the viral envelope. Previously,
we demonstrated that IFV decreased the infectability by damaging their constituent proteins prior to
nucleic acid degradation with photocatalysis reaction [31]. The photocatalytic action of TiO2 maypromote peroxidation of phospholipid components of the enveloped membrane, inducing significant
damage in membranes as mode of inactivation that is observed for bacteria. The phospholipid content
may influence the virucidal activity of photocatalysis. Further study is required to clarify themechanism of virucidal activity by evaluating other viruses. Although there was a difference in an
effect caused by IFV and FCV, TiO2 is a potential, environment-friendly antivirus agent.
3. Experimental Section
3.1. Bacteria Strains
Drug-resistant and clinically pathogenic strains of GPC and GNR were used as reference strains toevaluate the bactericidal activity by photocatalysis. They were donated from other researchers orpurchased from ATCC (American Type Culture Collection, Manassas, VA, USA). They included the
GPC strains MRSA ATCC 43300, VRE ATCC 51575, PRSP ATCC 700677, and the GNR strains
E. coli ATCC 8739, S. marcescens ATCC 13880, MDRA, MDRP, and NDM-1 producing K.
pneumoniae BAA-2146. MDRA and MDRP were isolated from a hospital in Japan and they wereidentified to be resistant to ciprofloxacin (minimum inhibitory concentration: MIC ≥ 4 mg/mL),
imipenem (MIC ≥ 16 μg/mL), and amikacin (MIC ≥ 64 μg/mL). The profiles of these strains are listed
in Table 2.
Table 2. Profiles of the organisms tested in this study.
Species

Structure

Types of Infections aE. coliS. marcescens
MDRA
MDRP
NDM-1 producing
K. pneumoniae

Gram-negative rod
Gram-negative rod
Gram-negative rod
Gram-negative rod

Diarrhea, UTI, food poisoning, sepsis
Nosocomial infections, Pneumonia, UTI, and wound infections
Nosocomial infections, Pneumonia, UTI, VAP
Nosocomial infections, Pneumonia, UTI, VAP, Bacteremia, cystic fibrosis

Gram-negative rod

Nosocomial infections, Pneumonia, UTI, Bacteremia

MRSA

Gram-positive cocci

VRE
PRSP
IFV
FCV

Gram-positive cocci
Gram-positive cocci
Enveloped virus
Non-enveloped virusaNosocomial infections, skin infections, food poisoning, pneumonia,
meningitis, bacteremia
Nosocomial infections, UTI, meningitis, bacteremia
Community-acquired pneumonia, bacteremia, bacterial meningitis
Respiratory tract infections
Respiratory infection in cats

UTI, Urinary tract infection; VAP, Ventilator Associated Pneumonia.

Catalysts 2013, 3

318

The strains except PRSP were cultivated twice consecutively on nutrient agar plates (Becton,
Dickinson and Company, Franklin Lakes, NJ, USA) while PRSP was cultivated on 5% sheep blood
agar plates at 37 °C for 18–20 h. Subsequently, the live cells were suspended in 1/500 nutrient brothsolutions, which contained 6 mg of meat extract, 20 mg of peptone, and 10 mg of NaCl per liter(pH 7.0), and then diluted to approximately 107 colony-forming units per milliliter. All reagents andconcentrations used were prepared in accordance with the JIS R 1702 standard method [47].
3.2. Virus Strains
The human IFV A/PR8/H1N1 strain and FCV strain F-9 ATCC VR-782 were used as reference
strains to evaluate the virucidal activity by photocatalysis. They were purchased from ATCC. IFV waspropagated by using 11-day-old embryonated chicken eggs and purified by using previously describedmethods [43]. FCV was propagated by using confluent layers of Crandell-Reese feline kidney (CRFK)
cell cultures. The harvested samples were clarified and concentrated by a combination of depth andmembrane filters (microfiltration with 0.45-μm polypropylene mesh and cross-flow polysulfone
hollow-fiber ultrafiltration with a 100-kDa cut-off, GE Healthcare, Japan) [48]. Subsequently, the virus
was purified by sucrose density gradient centrifugation using a linear sucrose gradient of
30%–70% [49]. Each individual experimental sample was adjusted to 0.1 mg/mL protein withphosphate-buffered saline, and 0.1 mg/mL of bovine serum albumin was added as stabilizing
agent [50,51]. The virus titer of IFV and FCV was determined by using the plaque technique on
confluent layers of Madin-Darby canine kidney (MDCK) cell or CRFK cell cultures grown in 12-wellculture plates, respectively, as described before[49,52].
3.3. TiO2-Coated Glass
TiO2-coated glass (T3 sample, 50 mm × 50 mm) was obtained from TOTO Ltd., (Fukuoka, Japan)
by employing a spin-coating process with TiO2 (obtained from Ishihara Sangyo Kaisha, Ltd., Osaka,
Japan) and heat treatment at 150 °C. As mentioned previously, the T3 sample was used as test sample
for the preparation of ISO27447:2009(E) [53] and JIS R 1702 [47] for evaluating the photocatalytic
antibacterial activity. The phase space of this sample was smooth, the thickness was approximately
200 nm, and the specific surface area was 300 m2/g. Non-TiO2 coated glass was used as the control.
The T3 sample and control glass plates were washed with sterilized water and then sterilized by UV
exposure overnight or wiped with ethanol.
3.4. Photocatalytic Reaction
The photocatalytic inactivation of bacteria and virus was investigated by applying the test method
for the evaluation of the antibacterial effect and the test method used in our previous study for theevaluation of the antivirus effect [31]. Overhead illumination by long-wavelength UV (UV-A) light
was provided by using tubular 20-W black light fluorescent lamps (FL20S Black Light Blue (BLB);λ = 352–368 nm; Toshiba, Japan). Illumination conditions of 0.1 mW/cm2 were adjusted by changing
the horizontal distance between the samples and lamp. UV-A intensity was analyzed by using an
integrating actinometer for photocatalysis (C9536–01/H9958; Hamamatsu Photonics Co. Ltd.,Catalysts 2013, 3319

Hamamatsu, Japan). All experiments were performed in a light-tight box to prevent any influence ofindoor light and sunlight.
The bacterial or viral suspension (100 μL) was dropped onto the T3 sample or control grass andequally distributed by covering it with adhesive film (VF-15, Kokuyo Co. Ltd., Osaka, Japan,
40 mm × 40 mm). The sample was then illuminated with UV-A irradiation. The photocatalytic
reaction utilized light intensity in the range of 0.1 mW/cm2 at the sample surface, and the UV-A
irradiation time was 0–8 h. After illumination for a certain period of time, the sample was immersed
and washed with 10 mL of soybean-casein digest broth, which contained lecithin and polysorbate. The
collected viable bacteria or virus cells were serially diluted in phosphate-buffered saline and evaluatedby using the colony formation method or plaque assay, respectively. All experiments were repeatedindependently more than three times, and the average titer was determined. Statistical analysis was
performed by the Bonferroni test using SPSS software (SPSS Inc., Chicago, IL, USA). p < 0.05 wasconsidered statistically significant. All reagents and concentrations used were prepared in accordancewith ISO27447:2009(E) [53] and JIS R 1702 [47], and with minor modifications for the concentrationsof viruses as previously reported [31].
4. Conclusions
We have demonstrated broad-spectrum biocide efficacy of photocatalysis against both bacteria(GPC and GNR, including drug-resistant bacteria) and viruses (enveloped and non-enveloped virus).
GPC such as MRSA and VRE were easily inactivated by photocatalysis, whereas some GNRs such as
E. coli and MDRP were gradually inactivated by the photocatalysis. The difference in the bactericidal
activity against GPC and GNR could be associated with the structure of their surface layer. IFV, anenveloped virus, was significantly faster inactivated by photocatalysis compared with FCV, anon-enveloped virus. These data indicated that the virucidal activity of photocatalysis may depend on
the presence or absence of a viral envelope.
The LRV of representative species induced in the presence of the tested photocatalyst were found to
decrease in the following order (LRV of the species at 4 h): MRSA (4.4) > IFV (3.6) > E. coli (2.1) >FCV (1.3). Photocatalysis has broad-spectrum microbicidal activity and the effects were found to be in
the following order: GPC > enveloped virus > GNR > non-enveloped virus. Although there was adifference in the effect due to the surface structure, TiO2 is a potential, environmentally friendly
microbicidal agent.
Photocatalysis would reduce the risk of contact infection spreading within the environmental
surfaces at facilities, e.g., as measure against healthcare-associated infections at hospitals or healthcare
facilities, and prevention of the spread of community-acquired infections at airports or day-care
centers. Furthermore, because of the high level and broad-spectrum biocide efficacy of photocatalysis,
it is considered a valuable biocide method for use in various areas, e.g., sewage disposal treatment
systems and air purification systems [54].
Acknowledgments
We would like to thank Ishihara Sangyo Kaisha, LTD for the gift of TiO2. We also thank TOTO Ltd.
for assistance in the preparation of the TiO2-coated samples. We thank Hiromi Yamamoto for excellent

Catalysts 2013, 3

320

technical assistance. The original studies in our program were supported by the New Energy andIndustrial Technology Development Organization (NEDO) of Japan.
Conflict of Interest
The authors declare no conflict of interest.
References
1.

Bratu, S.; Landman, D.; Haag, R.; Recco, R.; Eramo, A.; Alam, M.; Quale, J. Rapid spread ofcarbapenem-resistant Klebsiella pneumoniae in New York City: A new threat to our antibiotic
armamentarium. Arch. Int. Med. 2005, 165, 1430–1435.
2. Livermore, D.M. Fourteen years in resistance. Int. J. Antimicrob. Agents 2012, 39, 283–294.
3. Burke, J.P. Infection control—A problem for patient safety. N. Engl. J. Med. 2003, 348, 651–656.
4. Lankford, M.G.; Collins, S.; Youngberg, L.; Rooney, D.M.; Warren, J.R.; Noskin, G.A.
Assessment of materials commonly utilized in health care: Implications for bacterial survival andtransmission. Am. J. Infect. Control 2006, 34, 258–263.
5. Neely, A.N.; Maley, M.P. Survival of enterococci and staphylococci on hospital fabrics andplastic. J. Clin. Microbiol. 2000, 38, 724–726.
6. Kramer, A.; Schwebke, I.; Kampf, G. How long do nosocomial pathogens persist on inanimate
surfaces? A systematic review. BMC Infect. Dis. 2006, 6, 130.
7. Weber, D.J.; Rutala, W.A. Use of germicides in the home and the healthcare setting: Is there arelationship between germicide use and antibiotic resistance? Infect. Control Hosp. Epidemiol.
2006, 27, 1107–1119.
8. Ramirez, J.A.; Anzueto, A.R. Changing needs of community-acquired pneumonia. J. Antimicrob.
Chemother. 2011, 66, iii3–iii9.
9. Nordmann, P.; Naas, T.; Poirel, L. Global spread of carbapenemase-producing enterobacteriaceae.
Emerg. Infect. Dis. 2011, 17, 1791–1798.
10. Fraser, C.; Donnelly, C.A.; Cauchemez, S.; Hanage, W.P.; van Kerkhove, M.D.;
Hollingsworth, T.D.; Griffin, J.; Baggaley, R.F.; Jenkins, H.E.; Lyons, E.J.; et al. Pandemic
potential of a strain of influenza A (H1N1): Early findings. Science 2009, 324, 1557–1561.
11. Hutson, A.M.; Atmar, R.L.; Estes, M.K. Norovirus disease: Changing epidemiology and hostsusceptibility factors. Trends Microbiol. 2004, 12, 279–287.
12. Boone, S.A.; Gerba, C.P. Significance of fomites in the spread of respiratory and enteric viraldisease. Appl. Environ. Microbiol. 2007, 73, 1687–1696.
13. Sakaguchi, H.; Wada, K.; Kajioka, J.; Watanabe, M.; Nakano, R.; Hirose, T.; Ohta, H.;
Aizawa, Y. Maintenance of influenza virus infectivity on the surfaces of personal protective
equipment and clothing used in healthcare settings. Environ. Health Prev. Med. 2010, 15,
344–349.
14. Goldmann, D.A. Transmission of viral respiratory infections in the home. Pediatr. Infect. Dis. J.
2000, 19, S97–S102.

Catalysts 2013, 3

321

15. Wu, H.M.; Fornek, M.; Schwab, K.J.; Chapin, A.R.; Gibson, K.; Schwab, E.; Spencer, C.;
Henning, K. A norovirus outbreak at a long-term-care facility: The role of environmental surface
contamination. Infect. Control Hosp. Epidemiol. 2005, 26, 802–810.
16. Infection Control in Health Care Facilities. Available online: hiip://www.cdc.gov/
flu/professionals/infectioncontrol/ (accessed on 15 October 2012).
17. Nakano, R.; Nakano, A.; Abe, M.; Inoue, M.; Okamoto, R. Regional outbreak of CTX-M-2
beta-lactamase-producing Proteus mirabilis in Japan. J. Med. Microbiol. 2012, 61, 1727–1735.
18. Guidelines for Environmental Infection Control in Health-Care Facilities. Recommendations of
CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC); U.S.
Department of Health and Human Services Centres for Disease Control and Prevention (CDC):
Atlanta, GA, USA, 2003; pp. 1–42.
19. Hashimoto, K.; Irie, H.; Fujishima, A. TiO2 photocatalysis: A historical overview and future
prospects. Jpn. Soc. Appl. Phys. 2005, 44, 8269–8285.
20. Nakata, K.; Fujishima, A. TiO2 photocatalysis: Design and applications. J. Photochem. Photobiol.
C 2012, 13, 169–189.
21. Nakata, K.; Ochiai, T.; Murakami, T.; Fujishima, A. Photoenergy conversion with TiO2
photocatalysis: New materials and recent applications. Electrochim. Acta 2012, 84, 103–111.
22. Linsebigler, A.L.; Lu, G.; Yates, J.T. Photocatalysis on TiO2 surfaces: Principles, mechanisms,
and selected results. Chem. Rev. 1995, 95, 735–758.
23. Daimon, T.; Hirakawa, T.; Kitazawa, M.; Suetake, J.; Nosaka, Y. Formation of singlet molecularoxygen associated with the formation of superoxide radicals in aqueous suspensions of TiO2
photocatalysts. Appl. Catal. A 2008, 340, 169–175.
24. Ishibashi, K.I.; Fujishima, A.; Watanabe, T.; Hashimoto, K. Generation and deactivation
processes of superoxide formed on TiO2 film illuminated by very weak UV light in air or water.
J. Phys. Chem. B 2000, 104, 4934–4938.
25. Wei, C.; Lin, W.Y.; Zainal, Z.; Williams, N.E.; Zhu, K.; Kruzic, A.P.; Smith, R.L.;
Rajeshwar, K. Bactericidal activity of TiO2 photocatalyst in aqueous media: Toward
a solar-assisted water disinfection system. Environ. Sci. Technol. 1994, 28, 934–938.
26. Ryu, H.; Gerrity, D.; Crittenden, J.C.; Abbaszadegan, M. Photocatalytic inactivation of
cryptosporidium parvum with TiO2 and low-pressure ultraviolet irradiation. Water Res. 2008, 42,
1523–1530.
27. Ochiai, T.; Fujishima, A. Photoelectrochemical properties of TiO2 photocatalyst and its
applications for environmental purification. J. Photochem. Photobiol. C 2012, 13, 247–262.
28. Sunada, K.; Watanabe, T.; Hashimoto, K. Bactericidal activity of copper-deposited TiO2 thin film
under weak UV light illumination. Environ. Sci. Technol. 2003, 37, 4785–4789.
29. Mitoraj, D.; Janczyk, A.; Strus, M.; Kisch, H.; Stochel, G.; Heczko, P.B.; Macyk, W. Visible light
inactivation of bacteria and fungi by modified titanium dioxide. Photochem. Photobiol. Sci. 2007,
6, 642–628.
30. Fujishima, A.; Zhang, X.; Tryk, D. TiO2 photocatalysis and related surface phenomena. Surf. Sci.
Rep. 2008, 63, 515–582.

Catalysts 2013, 3322

31. Nakano, R.; Ishiguro, H.; Yao, Y.; Kajioka, J.; Fujishima, A.; Sunada, K.; Minoshima, M.;
Hashimoto, K.; Kubota, Y. Photocatalytic inactivation of influenza virus by titanium dioxide thinfilm. Photochem. Photobiol. Sci. 2012, 11, 1293–1298.
32. Ishiguro, H.; Nakano, R.; Yao, Y.; Kajioka, J.; Fujishima, A.; Sunada, K.; Minoshima, M.;
Hashimoto, K.; Kubota, Y. Photocatalytic inactivation of bacteriophages by TiO2-coated glass
plates under low-intensity, long-wavelength UV irradiation. Photochem. Photobiol. Sci 2011, 10,
1825–1829.
33. Ishiguro, H.; Yao, Y.; Nakano, R.; Hara, M.; Sunada, K.; Hashimoto, K.; Kajioka, J.;
Fujishima, A.; Kubota, Y. Photocatalytic activity of Cu2+/TiO2-coated cordierite foam inactivatesbacteriophages and Legionella pneumophila. Appl. Catal. B 2013, 129, 56–61.
34. Gerrity, D.; Ryu, H.; Crittenden, J.; Abbaszadegan, M. Photocatalytic inactivation of viruses
using titanium dioxide nanoparticles and low-pressure UV light. J. Environ. Sci. Health. A 2008,
43, 1261–1270.
35. Kavitha, T.; Gopalan, A.I.; Lee, K.P.; Park, S.Y. Glucose sensing, photocatalytic and antibacterial
properties of graphene-ZnO nanoparticle hybrids. Carbon 2012, 50, 2994–3000.
36. Synnott, D.W.; Seery, M.K.; Hinder, S.J.; Michlits, G.; Pillai, S.C. Anti-bacterial activity ofindoor-light activated photocatalysts. Appl. Catal. B 2013, 130–131, 106–111.
37. Nixdorff, K.; Gmeiner, J.; Martin, H.H. Interaction of lipopolysaccharide with detergents and its
possible role in the detergent resistance of the outer membrane of Gram-negative bacteria.
Biochim. Biophys. Acta 1978, 510, 87–98.
38. McDonnell, G.; Russell, A.D. Antiseptics and disinfectants: Activity, action, and resistance. Clin.
Microbiol. Rev. 1999, 12, 147–179.
39. Blake, D.M.; Maness, P.C.; Huang, Z.; Wolfrum, E.J.; Huang, J.; Jacoby, W.A. Application of thephotocatalytic chemistry of titanium dioxide to disinfection and the killing of cancer cells. Sep.
Purif. Rev. 1999, 28, 1–50.
40. Maness, P.C.; Smolinski, S.; Blake, D.M.; Huang, Z.; Wolfrum, E.J.; Jacoby, W.A. Bactericidal
activity of photocatalytic TiO2 reaction: Toward an understanding of its killing mechanism. Appl.
Environ. Microbiol. 1999, 65, 4094–4098.
41. Sunada, K.; Kikuchi, Y.; Hashimoto, K.; Fujishima, A. Bactericidal and detoxification effects ofTiO2 thin film photocatalysts. Environ. Sci. Technol. 1998, 32, 726–728.
42. Macnab, R.M. How bacteria assemble flagella. Annu. Rev. Microbiol. 2003, 57, 77–100.
43. Ibáñez, J.A.; Litter, M.I.; Pizarro, R.A. Photocatalytic bactericidal effect of TiO2 on Enterobactercloacae. J. Photochem. Photobiol. A 2003, 157, 81–85.
44. Horowitz, B.; Wiebe, M.E.; Lippin, A.; Stryker, M.H. Inactivation of viruses in labile blood
derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate detergent
combinations. Transfusion 1985, 25, 516–522.
45. Beekes, M.; Mielke, M.; Pauli, G.; Baier, M.; Kurth, R. Aspects of risk assessment and risk
management of nosocomial transmission of classical and variant Creutzfeldt-Jakob disease withspecial attention to German regulations. Contrib. Microbiol. 2004, 11, 117–135.
46. Rutala, W.A.; Weber, D.J.; Healthcare Infection Control Practices Advisory Committee(HICPAC). Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008; Centres
for Disease Control and Prevention (CDC): Atlanta, GA, USA, 2008.

Catalysts 2013, 3323

47. Shima, E.H. JIS R 1702, Fine Ceramics (Advanced Ceramics, Advanced Technical
Ceramics)—Test Method for Antibacterial Activity of Photocatalytic Products underPhotoirradiation and Efficacy; Japanese Standards Association: Tokyo, Japan, 2006.
48. Kalbfuss, B.; Genzel, Y.; Wolff, M.; Zimmermann, A.; Morenweiser, R.; Reichl, U. Harvesting
and concentration of human influenza A virus produced in serum-free mammalian cell culture forthe production of vaccines. Biotechnol. Bioeng. 2007, 97, 73–85.
49. Kreutz, L.C.; Seal, B.S.; Mengeling, W.L. Early interaction of feline calicivirus with cells in
culture. Arch. Virol. 1994, 136, 19–34.
50. Van Regenmortel, M.H.V.; Fauquet, C.M.; Bishop, D.H.L. Virus Taxonomy. Classification and
Nomenclature of Viruses; Academic Press: New York, NY, USA, 2000; pp. 835–849.
51. Yachida, S.; Aoyama, S.; Takahashi, N.; Iritani, Y.; Katagiri, K. Plastic multiwell plates to assay
avian infectious bronchitis virus in organ cultures of chicken embryo trachea. J. Clin. Microbiol.
1978, 8, 380–387.
52. Tobita, K.; Sugiura, A.; Enomote, C.; Furuyama, M. Plaque assay and primary isolation of
influenza A viruses in an established line of canine kidney cells (MDCK) in the presence oftrypsin. Med. Microbiol. Immunol. 1975, 162, 9–14.
53. Fine Ceramics (Advanced Ceramics, Advanced Technical Ceramics)—Test Method for
Antibacterial Activity of Semiconducting Photocatalytic Materials; Japan Industrial Standards
Committee: Tokyo, Japan, 2009.
54. Ochiai, T.; Nanba, H.; Nakagawa, T.; Masuko, K.; Nakata, K.; Murakami, T.; Nakano, R.;
Hara, M.; Koide, Y.; Suzuki, T.; et al. Development of an O3-assisted photocatalytic
water-purification unit by using a TiO2 modified titanium mesh filter. Catal. Sci. Technol. 2012,
2, 76.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license(http://creativecommons.org/licenses/by/3.0/).