Elsevier

Water Research

Volume 41, Issue 4, February 2007, Pages 842-852
Water Research

Disinfection of Legionella pneumophila by photocatalytic oxidation

https://doi.org/10.1016/j.watres.2006.11.033Get rights and content

Abstract

Photocatalytic oxidation (PCO) was proven to be efficacious in the inactivation of Legionella pneumophila serogroup 1 Strains 977, 1009, 1014 and ATCC 33153. The local (Strains 997, 1009 and 1014) and ATCC (Strain 33153) strains showed sensitivity differences towards PCO. The inactivation mechanisms of PCO were investigated by transmission and scanning electron microscopy by which PCO was found to disintegrate the cells eventually. Before the disintegration, there was lipid peroxidation of outer and cytoplasmic membrane causing holes formation and leading to the entry of radical dotOH into the cells to oxidize the intracellular components. Fatty acid profile analysis found that the amount of saturated, 16-carbon branched-chain fatty acid, which is predominant in Legionella, decreased in the surviving populations from PCO. A relationship between the amount of this fatty acid and the PCO sensitivity of the tested strains was also observed. Mineralization of cells by PCO was proven by total organic carbon analysis.

Introduction

Legionella species have been known to cause Legionnaires’ diseases (pneumonic legionellosis) and Pontiac fever (severe influenza-like illness) (Kim et al., 2002). Several reports have shown a clear association between the presence of Legionella in hot water systems and the occurrence of legionellosis (Aurell et al., 2004). Water systems such as industrial cooling towers (Ishimatsu et al., 2001), hospital hot-water distribution systems (Liu et al., 1995; Lin et al., 1998), shower, spa (Leoni et al., 2001) and hot spring water (Ohno et al., 2003) have been found to be the sources of the bacteria. Outbreaks of Legionnaires’ diseases have been reported in hospitals (Sabria and Yu, 2002; Stout and Yu, 2003) and industrial facilities (Ishimatsu et al., 2001; Moens et al., 2002). This led to the development of various preventive measures.

In 1997, the Hospital Infection Control Practices Advisory Committee of the Centers for Disease Control and Prevention (CDC) recommended only two disinfection modalities for controlling Legionella in hospital water systems: thermal eradication (superheating the water to 65 °C and flushing outlets) or hyperchlorination (1–2 mg/L) (Centers for Disease Control and Prevention, 1997). However, recolonization and safety concern were problems of these methods (Lin et al., 1998; Kim et al., 2002).

Photocatalysis by titanium dioxide (TiO2) could be an alternative or a complement to conventional water disinfection technologies. Photocatalytic oxidation (PCO) is one of the advanced oxidation processes (AOPs). Semi-conductor powder like TiO2 can be used as a photocatalyst. TiO2 photocatalyst has been extensively studied over the past 30 years for the removal of organic compounds from polluted water and air. When the TiO2 photocatalyst is irradiated with near ultraviolet (UV) light with wavelength (λ) shorter than 385 nm (UV-A), reactive oxygen species (ROS) such as hydroxyl radicals (radical dotOH), superoxide anions (radical dotO2) and hydrogen peroxide (H2O2) are generated. These ROS, especially the radical dotOH, are even more reactive and oxidizing than chlorine (Bull and Zeff, 1992). Organic pollutants and bacteria sorbed on the TiO2 particles surface will then be oxidized by the radical dotOH generated (Rincón and Pulgarin, 2003, Rincón and Pulgarin, 2004). The radical dotOH is highly effective for both the oxidation of organic substances and inactivation of bacteria and virus. Photocatalytic inactivation of bacteria and yeasts including Escherichia coli (Matsunaga et al., 1988; Ireland et al., 1993; Melián et al., 2000; Salih, 2002; Lu et al., 2003; Rincón and Pulgarin, 2003, Rincón and Pulgarin, 2004), Candida albicans, Enterococcus faecium, Pseudomonas aeruginosa and Staphylococcus aureus (Kühn et al., 2003); Streptoccocus faecalis (Melián et al., 2000), Streptoccocus mutans (Saito et al., 1992), Lactobacillus acidophilus and Saccharomyces cerevisiae (Matsunaga et al., 1985) as well as poliovirus (Watts et al., 1995) have been reported.

As Legionella spp. are sensitive to relatively low levels of H2O2 and radical dotO2, which are produced in the medium especially after exposure to light (Hoffman et al., 1983), PCO should have certain effect on the viability of the bacteria. As early as in 1985, Matsunaga et al. (1985) proposed that the fundamental reason for the cells killed by PCO was the direct photochemical oxidation of the intracellular coenzyme A (CoA) caused the formation of dimers and resulted in a decrease in respiratory activities (Matsunaga et al., 1985, Matsunaga et al., 1988). Saito et al. (1992) found the rapid leakage of potassium ions from the TiO2-treated cells along with the decrease in the cell viability. Also, proteins and RNA were shown released slowly from the cells upon a longer reaction time. From such results, they concluded that there should be a significant disorder in the cell membranes and eventually the cell walls were decomposed (Saito et al., 1992). Sunada et al. (1998) found the degradation of endotoxin which is a lipopolysaccharide (LPS) cell wall (outer membrane) constituent of Gram-negative bacteria. Maness et al. (1999) suggested TiO2 photocatalysis promotes peroxidation of the polyunsaturated phospholipid component of the lipid membrane and induced major disorder in the cell membrane. This causes the loss of essential membrane-bound functions such as respiratory activities of the bacteria and led to cell death.

Recently, Kühn et al. (2003) used light and scanning electron microscopy (SEM) to examine the cell surfaces of C. albicans and found that the radical dotOH had led to direct damage to cell wall. Moreover, using SEM, Jacoby et al. (1998) were able to show that E. coli on TiO2-coated glass slides irradiated for 75 h were completely destroyed and removed by complete mineralization. Apart from SEM, transmission electron microscopy (TEM) was carried out by Saito et al. (1992) to show the broken cell walls of Streptococcus sobrinus after photocatalysis. Lu et al. (2003) applied atomic force microscopy (AFM) imaging and fluorescence measurements of quantum dots (QDs) entry to the cells to prove the immediate decomposition of the cell wall and a further damage of the cell membrane.

One of the key issues for implementing the measure is the selection of disinfectant(s) and optimal conditions for its use. In the present study, PCO was used to disinfect Legionella pneumophila in the aqueous medium and the process was optimized. As 70–90% of all culture-confirmed or urine antigen-confirmed cases are caused by L. pneumophila serogroup 1 (Marston et al., 1994; Benin et al., 2002), it was chosen for this study. The disinfection mechanism(s) were also studied in order to facilitate the enhancement of the efficiency of the selected method.

Section snippets

Culture of microorganisms

L. pneumophila serogroup 1 Strains ATCC 33153, 977, 1009 and 1014 were used. Except the ATCC strain, all others were isolated from local water towers by the Microbiology Department, The Chinese University of Hong Kong. In the present study, an active L. pneumophila culture was inoculated onto buffered charcoal yeast extract agar supplemented with α-ketoglutarate (BCYEα agar, Oxoid Limited, UK) and incubated under 5% CO2 for 3 days at 37 °C. The colonies were later aseptically washed by

Effect of PCO on cell viability

L. pneumophila serogroup 1 ATCC 33153 was shown to be UV365nm resistant. Preliminary PCO tests on the ATCC 33153 strain (with initial cell concentration of 107 cfu/mL) using 1000 mg/L of TiO2 and 108 μW/cm2 of UV365nm showed 4.5 log-reduction in the viable cell count after 90 min of PCO treatment. All the three controls showed no obvious reduction in the viable counts compared with the treatments. Such results showed that PCO was able to inactivate L. pneumophila. For the surviving colonies, they

Discussion

The preliminary PCO tests showed that PCO was able to reduce the viability of L. pneumophila. Therefore, PCO can be applied as an alternative to the conventional disinfection methods. For the surviving populations, their colonies were smaller in size. This indicated that they became weaker (with a lower growth rate) due to damages caused by PCO. Other than the size, the colonies formed were observed to be duller than the original one. To get a better understanding for this, fatty acid profile

Conclusions

  • (1)

    PCO was employed to inactivate the cells of four L. pneumophila serogroup 1 strain (Strain 977, Strain 1009, Strain 1014 and ATCC 33153) collected form different origins. The ATCC strain is less susceptible to PCO inactivation than other strains isolated locally.

  • (2)

    TEM and SEM studies indicate that the outer and cell membranes are the primary target site for PCO inactivation. Lipid peroxidation of these membranes by PCO plays important role in the first phase of inactivation.

  • (3)

    Fatty acid profiles of

Acknowledgments

The project was supported by research grants of Research Grant Council, Hong Kong SAR Government, allocated to P.K. Wong and C.Y. Chan. We would like to express our appreciation for the kind assistance from Professor David Yew of Department of Anatomy, The Chinese University of Hong Kong on the scanning electron micrographic analysis of the samples.

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