Current Microbiology

, Volume 52, Issue 1, pp 1–5

The Phototoxicity of Xanthene Derivatives Against Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae

Authors

    • School of Environmental Science and EngineeringShanghai Jiao Tong University
  • Lei Lu
    • School of Environmental Science and EngineeringShanghai Jiao Tong University
  • Shiyun Zhu
    • School of Environmental Science and EngineeringShanghai Jiao Tong University
  • Yahong Li
    • School of Environmental Science and EngineeringShanghai Jiao Tong University
  • Weimin Cai
    • School of Environmental Science and EngineeringShanghai Jiao Tong University
Article

DOI: 10.1007/s00284-005-0040-z

Cite this article as:
Wang, H., Lu, L., Zhu, S. et al. Curr Microbiol (2006) 52: 1. doi:10.1007/s00284-005-0040-z

Abstract

We assessed the phototoxicity of a series of xanthene derivatives against E. coli, S. aureus, and S. cerevisiae, measured the physicochemical properties of the photosensitizers, and found the relationship between them. Without illumination, the dyes tested showed almost the same level of inherent toxicity to the same organism, which showed the inherent toxicity of dyes was primarily dependent on the structure of parent molecule. Upon illumination, the photosensitizers showed obvious phototoxicity to all organisms. The dyes showed stronger phototoxicity to Gram-positive bacteria. With the increasing number of halogen substituents, the singlet oxygen yields increased and the phototoxic activity increased too. There was no obvious correlation between relative lipophilicity and activity in the current study. Our results showed xanthenes had the potential to act as alternatives to conventional antimicrobial compounds and also could be used for the decontamination of microbially polluted waters.

The research field of antimicrobial compounds is one of the current constant challenges. Unfortunately, an unpleasant cycle has recently appeared: as soon as a new drug is introduced, the strains resistant to that drug emerge [14]. There is an urgent need for some new drugs with novel mechanisms of attack.

Extensive research had been carried out to screen the photosensitizers with antimicrobial properties and illuminate their photodynamic mechanisms [2, 3, 19, 20]. Photosensitizers can be electronically excited by irradiation with light at the wavelength appropriate for that photosensitizer after being localized in the target organism [4]. Then the molecule will pass its excitation energy onto other biomolecules by two mechanisms. In the type I process, energy can be transferred between the excited photosensitizer and nearby biomolecules, yielding oxygenated free radicals; In the type II process, energy can be transferred between the excited photosensitizer and molecular oxygen, yielding singlet oxygen, 1O2 [6]. Such highly reactive products are able to photomodify some biomolecules in cells such as lipids, enzymes, and DNA [7, 11, 12], which will be lethal to cells. The non-specific nature of these attacking modes makes it unlikely for the bacteria to acquire the resistance to the photosensitizers.

Many photosensitizers had been shown to possess antimicrobial properties [20]. Xanthene derivatives were a class of photosensitizing molecules and some of them had been developed for commercial use as pesticides [5, 9]. In the present study, the phototoxicity of a series of xanthene derivatives against E. coli, S. aureus, and S. cerevisiae, was assessed.

Materials and Methods

Photosensitizers

All chemicals used in our experiments were of analytical grade. Fluorescein (Fl)-derived photosensitizers (Table 1) were purchased from Shanghai Chemical Regents Company (China). Na2Fl, Na2FlBr4, Na2FlI4, and Na2FlBr4Cl4 were stored as aqueous stock solutions (1 mM) at 4°C. FlBr2 and FlI2 were stored as 95% ethanol stock solutions (1 mM) at 4°C.
Table 1

Structures of photosensitizers

https://static-content.springer.com/image/art%3A10.1007%2Fs00284-005-0040-z/MediaObjects/284_2005_40_t1.gif

Photosensitizer

X

Y

Fluorescein, disodium salt (Na2Fl)

H

H

H

Dibromofluorescein (FlBr2)

Br

H

H

Diiodofluorescein (FlI2)

I

H

H

Tetrabromofluorescein. disodium salt (Na2FlBr4)

Br

Br

H

Tetraiodofluorecein. disodium salt (Na2FlI4)

I

I

H

Tetrachlorotetrabromofluorescein. disodium salt (Na2FlBr4Cl4)

Br

Br

Cl

Light source

150-W metal halide lamp (Philips, The Netherlands), giving a light fluence of 15 mW cm−2, was used in toxicity tests. The fluence of the polychromatic light was measured with a FZ-A light meter (Handy, China).

Physicochemical properities

The lipophilicities of the photosen- sitizers were calculated in terms of log P, the logarithm of their partition coefficients between 1-octanol and deionized water. Maximal dye absorption in the range of visible light was determined in aqueous solutions by a Unico 2102 UV/Vis spectrophotometer (Japan). The photosensitizing efficiency was measured as relative singlet oxygen yield (Φ′). According to the method described by Kraljic and Mohsni [13], the system imidazole plus p-nitrosodimethylaniline (RNO) can be used as a sensitive and selective test for the presence of 1O2 in aqueous solutions. The test is based on secondary bleaching of RNO as induced by the reaction of 1O2 with imidazole. In the present study, the solutions of different photosensitizers were irradiated in closed absorption cells of 10-mm light path for 5 min and the O.D. was determined before and after irradiation at 440 nm, the maximal absorption of RNO. The concentrations of photosensitizer, RNO, and imidazole were set to 30 μM, 50 μM and 8 mM, respectively. The experiments were carried out at room temperature (25°C). We set Φ′=1 for Na2FlBr4Cl4.

Bacterial strains and growth condition

E. coli (AS 1.129) and S. aureus (AS 1.72) were grown aerobically in liquid LB medium for approximately 18 h using an orbital incubator at 37°C, which corresponded to the cell concentration of around 2 × 109 cfu ml−1. S. cerevisiae (AS 2.101) was grown in liquid PDA medium at 28°C for approximately 24 h, which corresponded to around 2 × 108 cfu ml−1. For the phototoxicity tests, cells were diluted with fresh medium to a final cell concentration of 105 cfu ml−1.

Toxicity tests

Qualitative phototoxicity tests were carried out using the methods modified from Daniels [1]. Photosensitizers were dissolved in corresponding solvents to the concentration of 2 mg/mL. Five microliters of each solution was applied to a piece of filter paper disc (7-mm diameter, 10 μg/disc) and solvents were allowed to evaporate in the dark. Forty microliters of cells at the concentration of 105 cfu ml−1 was spread onto a plate evenly with a sterile bent glass rod. The discs were placed onto the freshly inoculated plates and all plates were incubated in dark for 30 min. Then, after 30-min irradiation period under the light fluence of 15 mW cm−2, all plates were incubated in dark again at the temperature mentioned above for another 48 h. The zones of inhibition were measured. For the dark control plates, we omitted the 30-min irradiation period. All assays were repeated 3 times and the results combined.

Toxicity tests were conducted as 10-fold replicates using 300-μL flat-bottomed 96-well micro-titre plates (Costar). Fresh cell suspensions were prepared as described above. These suspensions were then mixed with the chemicals shown in Table 1 to obtain twofold dilution series of each photosensitizer with the final concentrations varying from 1 to 500 μM. Two hundred microliters of these mixtures were then added to the wells of micro-titre plates and all plates were incubated in dark for 30 min. Then, after a 30-min irradiation period under the light fluence of 15 mW cm−2, all plates were placed in an incubator again in dark at the temperature mentioned above for 18 h. The resulting 10-μL cultures were streaked onto agar medium plates and incubated for another 18 h. After incubation, tested plates were examined for bacterial growth and the lowest concentration at which no colonies were observed was taken as the minimum lethal concentration (MLC) of a given photosensitizer. The experiments were repeated until the same results appeared.

Results and Discussion

The MLCs of Na2Fl, FlBr2, FlI2, Na2FlBr4, Na2FlI4, and Na2FlBr4Cl4 (structures shown in Table 1) were determined when directly against three microorganisms with or without illumination (Table 2). The results were consistent with the qualitative phototoxicity assays (Table 3). The spectral output of the light source coincided with the max absorption wavelength of the photosensitizers. Control experiments showed that in the absence of photosensitizers, illumination alone or ethanol-added medium had no effect on all organisms (data not shown).
Table 2

Toxicity of photosensitizers against E. coil, S. aureus, and S. cerevisiae

 

MLC (μM)

 

E. coli

S. aureus

S. cerevisiae

Photosensitizers

Dark

Light

Dark/Light

Dark

Light

Dark/Light

Dark

Light

Dark/Light

Na2Fl

 

 

15.6

 

FlBr2

500

250

2

125

31.3

4

250

3.9

64

FlI2

500

125

4

125

31.3

4

250

3.9

64

Na2FlBr4

500

125

4

125

2

62.5

250

2

125

Na2FlI4

500

125

4

62.5

1

62.5

250

1

250

Na2FlBr4Cl4

500

125

4

62.5

1

62.5

125

1

125

− = no obvious effects under the highest concentration.

Table 3

Qualitative phototoxicity assays

 

E. coli

S. aureus

S. cerevisiae

Photosensitizers

Dark

Light

Dark

Light

Dark

Light

Na2Fl

+

FlBr2

+

+

+

++

FlI2

+

+

+

++

Na2FlBr4

+

+

++

++

Na2FlI4

+

+

++

++

Na2FlBr4Cl4

+

+

++

++

Clear zone diameter + = 1–5 cm; ++ = 5–10 cm; − = no obvious effects.

Without illumination, Na2Fl showed no inherent toxicity to three organisms under the concentration as high as 500 μM. The inherent MLCs of the other photosensitizers to E. coli, S. aureus, and S. cerevisiae were 500 μM, 62.5 to 125 μM, and 125 to 250 μM, respectively. In the present study, all photosensitizers showed almost the same level of inherent toxicity to the same organism. The present study showed that the inherent toxicity was primarily dependent on the structure of parent molecule, and substituted groups had slight effects on the inherent activity.

Upon illumination, the photosensitizers showed obvious phototoxicity against either bacterium under the conditions in our experiments. The xanthenes showed stronger phototoxicity to Gram-positive bacteria. S. cerevisiae was found to be much more sensitive than bacteria to the same xanthene. Na2FlI4 and Na2FlBr4Cl4 were the most phototoxic dyes tested with the MLCs of 125, 1, and 1 μM when directly against E. coli, S. aureus, and S. cerevisiae, respectively. The physicochemical properities of the photosensitizers used in the present study are given in Table 4. In vitro chemical tests showed that each of the xanthene derivatives was able to photosensitize the production of singlet oxygen, in the order of Na2FlBr4Cl4 > Na2FlI4 > Na2FlBr4 > FlI2 > FlBr2 > Na2Fl. All dyes tested except Na2Fl were phototoxic to three organisms and generated significant levels of singlet oxygen. It is suggested that type II mechanism of photosensitization played an important role in such actions. With the increasing number of halogen substituents, the singlet oxygen yields increased and the phototoxic activity increased too. Some research has shown that the presence of heavy bromine or iodine atoms enhanced the yields of intersystem crossing to the reactive triplet state of the xanthene dyes [11].
Table 4

Physicochemical properties of photosensitizers

Photosensitizer

λmax (nm)a

Log P

Φ′b

Na2Fl

490

−0.28

0.02

FlBr2

504

1.01

0.67

FlI2

507

1.21

0.69

Na2FlBr4

517

−0.25

0.81

Na2FlI4

527

−0.24

0.92

Na2FlBr4Cl4

539

−0.21

1

a Measured in water.

b Relative to the singlet oxygen yield of Na2FlBr4Cl4.

The Log P values measured in the experiments showed that FlBr2 and FlI2 were lipophilic (Log P > 0), while Na2Fl, Na2FlBr4, Na2FlI4, and Na2FlBr4Cl4 were hydrophilic (Log P < 0). Variation in Log P was expected to affect the uptake and localization of the photosensitizers. In the current study, there was no obvious correlation between relative lipophilicity and activity. Due to the work of Pooler and Valenzeno [18], we knew that xanthenes were typically localized in cell membrane. At a molecular level, xanthenes mostly photosensitized the cross-linking of proteins and formed the hydroperoxides from unsaturated lipids, thereby increasing the osmotic fragility of the cells. It was not necessary for such photosensitizers to pass through the membrane to attack intracellular targets. However, Pimprikar and Heitz [17] observed the toxicity ratio to Aedes mosquito larvae ranged up to 2 orders of magnitude between the soluble and insoluble forms of the same xanthene dye. With the lipophilic xanthenes, mosquito larvae were able to filter feed on dye particles and thereby received a higher level of the dye. Thus, lipophilic xanthenes showed higher activity against mosquito larvae than organisms in present tests.

There was a clear tendency that xanthenes showed higher activity against Gram-positive bacteria. The additional layer of protection provided by the outer membrane of Gram-negative bacteria could generally hinder the binding of the photosensitizers and intercept photo-generated reactive species. Researchers drew the same conclusion in a previous study [16].

In the present study, photosensitizers showed relatively higher toxicity upon illumination and lower toxicity without illumination against yeast S. cerevisiae than bacteria. Yeast was shown to be more sensitive to phototoxic reaction. Thus, it may be considered that S. cerevisiae is better than E. coli or S. aureus in screening useful photosensitizers.

Our results showed the dyes tested here could kill bacteria and yeast at micromolar concentrations. In relation to therapeutic use, although several photosensitizers have been used successfully in phototherapy, the dyes tested here still need some possible structural modifications to make their λmax lie within the window of 600–900 nm used for the treatment of human conditions. All the tested dyes will not persist or remain toxic for a very long time in the environment. Previous research showed that exposure of Na2Fl or Na2FlBr4Cl4 to sunlight was expected to lead to photodegradation with a half time of approximately 1 hour [10]. Thus, Suredye (69% Na2FlBr4Cl4 and 31% Na2Fl by weight of active components) becomes the most successfully used photoinsecticide to control the fruit fly [8]. In addition, Na2FlBr4Cl4 had been approved by EPA (USA) for trial applications to control corn root worms in 2000 and some more of these dyes were in experiments for insect control. At the same time, some researchers stated the photosensitized inactivation of yeast and bacteria could be used for the decontamination of microbially polluted waters [15]. Thus, photoactivated dyes show potential as antimicrobial agents; additional studies are needed to define and explore their applications for biological control and decontamination.

Acknowledgments

We thank Dr. Xiaofan Zhang for his technical assistance.

Copyright information

© Springer Science+Business Media, Inc. 2006