Low intensity red laser action on Escherichia coli cultures submitted to stress conditions

J N Santos; C Roos; L L Barboza; F Paoli; A S Fonseca

doi:10.1088/1054-660X/24/12/125603

1. Introduction

Since Maiman manufactured the first, lasers have been considered for treatment of diseases in different biological tissues. Initially, high-intensity lasers were used for tissue destruction or ablation, but as from the second half of the 20th century, low intensity lasers (He–Ne laser) also are used in clinical protocols to treat diseases in soft tissues. Therapy based on low intensity lasers is used to treat wound healing [1], muscle repair [2], and reduce pain and inflammation [3]. Photodynamic therapy is another useful application of low intensity lasers [4]. These applications of low intensity red and near infrared lasers (600–1300 nm) increased widely when diode lasers became commercially available.

Clinical protocols based on low intensity lasers are justified mainly by biostimulation or biomodulation effect [5]. In fact, cell proliferation and survival, and ATP, nucleic acids and protein synthesis are increased after low intensity laser exposure [6, 7]. Also, laser irradiation alters gene expression in cell culture [8] and tissues [9].

Although mitochondrial components of the respiratory chain (cytochrome c, for example) are considered the effective chromophores to low intensity laser radiations in the therapeutic window, there are doubts whether indirect effects of these radiations could occur on other molecules (DNA, for example). It was suggested that low intensity lasers induce mitochondrial retrograde signalling with participation of a free radical [10]. Doubts about indirect effects of low intensity lasers seem to emerge from this point and it was suggested that DNA damage was caused by the free radical induced by laser exposure [11]. Other authors proposed also that laser-induced effects occur by photochemical mechanisms [12]. Moreover, some studies demonstrated that previous exposure to low intensity lasers protects cells against harmful radiations [10, 13–15] and chemical compounds [16]. In addition, Kujawa et al [17] did not find DNA damage in B14 cells exposed to low intensity infrared laser, while Mbene et al [18] reported DNA damage in human skin fibroblast cells induced by a red laser. It is possible that controversy whether low intensity lasers cause DNA lesions is due to different laser device parameters (wavelength, fluence, power, emission mode), but also due to differences in cell culture conditions and the cell line exposed to laser.

In fact, some studies have suggested that therapeutic effects of low intensity lasers at low fluences depend on the genetic characteristics and physiologic condition of cells [16, 19–23]. Also, laser-induced positive effects on biological tissues could occur and be measurable whether there is a pathologic process in course or whether the cells are under stressful conditions [7, 24, 25].

Escherichia coli (E. coli) cells presenting mutations in genes related to the repair of DNA lesions have been successfully used to demonstrate genotoxic effects of chemical [16] and physical agents [26]. For this, bacterial survival and filamentation induction are the biological endpoints accessed. Under stressful conditions, E. coli cells proficient and deficient in DNA repair mechanisms could respond differently to agents that disturb the DNA molecule.

Thus, the objective of this work was to evaluate the survival, filamentation and morphology of E. coli cultures proficient and deficient in the repair of oxidative DNA lesions exposed to low intensity red laser under hyposmotic stress.

2. Materials and methods

2.1. Low intensity red laser

Low intensity red laser (AlGaInP, 10 mW) used in this study, with emission in 658 nm, was purchased from HTM Eletrônica (São Paulo, Brazil).

2.2. Bacterial survival assay

Survival of E. coli AB1157 (wild type) and JW1625-1 (endonuclease III deficient) cultures exposed to low intensity red laser was evaluated under hyposmotic stress. From stocks in stationary growth phase, cultures of these strains were prepared for attaining stationary growth phase (10¹⁰ cells ml⁻¹, 18–20 h, 37 °C). Aliquots from these stationary cultures were taken and further incubated in a nutritive medium to reach exponential growth (10⁸ cells ml⁻¹). After that, the cells were collected by centrifugation (700 × g, 15 min) washed twice in saline at different NaCl concentrations (0.00, 0.30, 0.60 and 0.90% in distilled water) and suspended again in the respective saline concentration. Next, aliquots (100 µl, n = 5, for each fluence) of bacterial suspensions (10⁸ cells ml⁻¹) were exposed, at room temperature and under the white light (fluorescent lamps), to low intensity red laser at different fluences (1 and 8 J cm⁻²), in continuous-wave (power output of 10 mW, power density of 79.6 mW cm⁻²), with the laser source at 3.0 cm from the surface of bacterial suspension aliquots (distance top-bottom of a microcentrifuge flex tube). Exposure time of the aliquots was automatically adjusted by the laser device as a dose control. After laser exposure, bacterial suspensions were incubated at 37 °C for 20 min. Bacterial suspensions not exposed to laser were used as controls. Aliquots (10 µ) of bacterial suspensions were diluted in saline and spread onto Petri dishes containing solidified rich medium (1.5% agar). Colonies formed after overnight incubation at 37 °C were counted and the survival fraction was calculated by ratio between the number of viable cells after laser exposure (for each fluence) and the number of viable cells before laser exposure. Experiments were carried out in triplicate and the results are the average mean of three independent assays.

2.3. Bacterial filamentation assay and morphological analysis

To evaluate filamentation induction, exponential E. coli AB1157 and JW1625-1 cultures were obtained and exposed to low intensity red laser as described in the bacterial survival assay. Bacterial suspensions not exposed to laser were used as controls. After laser exposure and incubation (37 °C, 20 min), aliquots (20 µl) were withdrawn, spread onto microscopic slides and stained by the Gram method [27]. Bacterial cells were visualized by light microscopy (40 × magnification), photographed to determine the percentage of bacterial filamentation, as well as to measure area and perimeter by Image ProPlus software. Experiments were carried out in duplicate and the results are represented as the mean of two independent assays.

2.4. Statistical analysis

Data are reported as means and standard deviation (means ± SD) of the survival fraction, percentages of bacterial filaments, area and perimeter. The two-way analysis of variance (ANOVA) test was performed to verify possible statistical differences followed by Bonferroni post-test with p < 0.05 as the less significant level. InStat Graphpad software was used to perform statistical analysis (GraphPad InStat for Windows XP, GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Survival of E. coli cultures exposed to laser under hyposmotic stress

Figure 1 shows the survival fractions of E. coli AB1157 exposed to low intensity red laser at different fluences in low saline concentrations (hyposmotic stress). Data in this figure shows that survival fractions of bacterial suspensions not exposed to laser are not significantly (p > 0.05) altered by hyposmotic stress when compared to bacterial suspensions in physiologic NaCl concentration (0.9% NaCl). However, survival fractions of bacterial suspensions exposed to red laser at the lower fluence (1 J cm⁻²) are significantly (p < 0.05) increased in 0.6% NaCl when compared to survival fractions of bacterial suspensions in 0.9% NaCl or when compared to bacterial suspensions not exposed to laser in 0.6% NaCl. Also, data in figure 1 show that survival fractions of bacterial suspensions exposed to red laser at the higher fluence (8 J cm⁻²) in the higher hyposmotic stress (0.0% NaCl) are significantly (p < 0.05) decreased when compared to bacterial suspensions not exposed to laser in the same conditions.

Effects of low intensity red laser were also evaluated on the survival of endonuclease III deficient E. coli cultures (JW1625-1) submitted to hyposmotic stress (figure 2). Similar to those obtained with wild type E. coli strain (AB1157), survival fractions of endonuclease III deficient bacterial suspensions not exposed to laser in hyposmotic stress are not significantly (p > 0.05) modified when compared to bacterial suspension in 0.9% NaCl. However, survival fractions of bacterial suspensions exposed to low intensity red laser are significantly (p < 0.05) decreased in 0.6% NaCl when compared to survival fractions of bacterial suspensions exposed to the same laser fluences in 0.9% NaCl or when compared to survival fraction of bacterial suspension not exposed to laser.

**Figure 2.** Bacterial survival fractions of *E. coli* JW1625-1 exposed to low intensity red laser under hyposmotic stress. Bacterial suspensions in exponential growth phase were exposed to laser (1 and 8 J cm⁻²) and incubated in NaCl solutions of different concentrations (37 °C, 20 min). After that, aliquots of these bacterial suspensions were diluted, spread onto Petri dishes containing nutritive medium, incubated (37 °C, 18 h) and the colony-forming units were counted to calculate the survival fractions. (*) p < 0.05 when compared to bacterial survival fraction in 0.9% NaCl solution, (#) p < 0.05 when compared to bacterial survival fraction of cells not exposed to laser.
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3.2. Bacterial filamentation in E. coli cultures exposed to laser under hyposmotic stress

To evaluate the effects of hyposmotic stress on bacterial filamentation induction, E. coli AB1157 cultures were exposed to low intensity red laser at different NaCl concentrations. Figure 3 shows the percentage of bacterial filaments in E. coli AB1157 suspensions after exposure to low intensity red laser in different NaCl concentrations. Data in this figure indicate that, in bacterial suspensions not exposed to laser, incubation in low NaCl concentrations significantly (p < 0.05) increases the percentage of bacterial filaments when compared to the percentage of bacterial filaments in bacterial suspension in NaCl 0.9%. However, exposure to low intensity red laser significantly (p < 0.05) decreases the percentages of bacterial filaments in bacterial suspensions in low NaCl concentrations when compared to bacterial suspensions not exposed to laser or bacterial suspensions in 0.9% NaCl, except in 0.0% NaCl. Also, data in figure 3 shows that laser exposure significantly (p < 0.05) increases the percentages of bacterial filaments in 0.9% NaCl.

**Figure 3.** Percentages of bacterial filaments in *E. coli* AB1157 exposed to low intensity red laser under hyposmotic stress. Bacterial suspensions in exponential growth phase were exposed to laser (1 and 8 J cm⁻²) and incubated in NaCl solutions of different concentrations (37 °C, 20 min). After that, aliquots of these bacterial suspensions were spread onto microscopic slides, stained by Gram method, visualized by light microscopy, bacterial filaments were analysed by Image ProPlus software and percentages of bacterial filaments were calculated. (*) p < 0.05 when compared to percentage of bacterial filaments in 0.9% NaCl solution, (#) p < 0.05 when compared to percentage of bacterial filaments in bacterial suspensions not exposed to laser.
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To evaluate whether bacterial filamentation induction depends on DNA repair mechanisms and hyposmotic stress, E. coli JW1625-1 cultures were exposed to low intensity red laser at different NaCl concentrations (figure 4). Different from wild type cultures (AB1157), bacterial suspensions not exposed to laser, or exposed to laser, under hyposmotic stress present the significantly (p < 0.05) lowest percentages of bacterial filament when compared to percentages of bacterial filaments in bacterial suspensions in 0.9% NaCl. In bacterial suspensions in 0.9% NaCl, exposure to low intensity red laser significantly (p < 0.05) increases the percentages of bacterial filaments. Also, exposure to laser at the higher fluence (8 J cm⁻²) increases the percentage of bacterial filaments in bacterial suspensions in 0.6% NaCl when compared to the percentage of bacterial filaments in bacterial suspensions not exposed to laser.

**Figure 4.** Percentages of bacterial filaments in *E. coli* JW1625-1 exposed to low intensity red laser under hyposmotic stress. Bacterial suspensions in exponential growth phase were exposed to laser (1 and 8 J cm⁻²) and incubated in NaCl solutions of different concentrations (37 °C, 20 min). After that, aliquots of these bacterial suspensions were spread onto microscopic slides, stained by Gram method, visualized by light microscopy, bacterial filaments were analysed by Image ProPlus software and percentages of bacterial filaments were calculated. (*) p < 0.05 when compared to percentage of bacterial filaments in 0.9% NaCl solution, (#) p < 0.05 when compared to percentage of bacterial filaments in bacterial suspensions not exposed to laser.
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3.3. Morphology of E. coli cells exposed to low intensity red laser under hyposmotic stress

Morphological analysis was accessed by area and perimeter measurements of cells from bacterial suspensions exposed to low intensity red laser under hyposmotic stress. In figure 5, data show that area values of wild type E. coli AB1157 cells not exposed to laser in low NaCl concentrations are not significantly (p > 0.05) different from the area value of cells in 0.9% NaCl. Similarly, there is no significant difference between areas of cells exposed to red laser at the lower fluence (1 J cm⁻²) in low NaCl and in 0.9% NaCl, except for cells in the higher hyposmotic stress (0.0% NaCl) when compared to the area of cells in 0.9% NaCl. However, when the area of these cells is compared to the area of cells not exposed to laser, there is significant (p < 0.05) difference, except for cells in 0.9% NaCl. Also, data in figure 5 show that the area of cells exposed to laser at the higher fluence in 0.6% NaCl is significantly (p < 0.05) different from the area of cells in 0.9% and different from the area of cells not exposed to laser in 0.6% NaCl.

**Figure 5.** Area of *E. coli* AB1157 cells exposed to low intensity red laser under hyposmotic stress. *E. coli* AB1157 cultures in exponential growth phase were exposed to low intensity red laser (1 and 8 J cm⁻²) and incubated in NaCl solutions of different concentrations (37 °C, 20 min). After that, aliquots of these bacterial suspensions were spread onto microscopic slides, stained by Gram method, visualized by light microscopy and analysed by Image ProPlus software to measure the area of bacterial cells. (*) p < 0.05 when compared to area of bacterial cells in 0.9% NaCl solution, (#) p < 0.05 when compared to area of bacterial cells not exposed to laser.
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To confirm the area measurement data, the perimeters of wild type E. coli cells were also measured (figure 6). Similarly to the results observed with area of cells, data in this figure show that perimeters of cells not exposed to laser are not significantly (p > 0.05) modified by hyposmotic stress. Laser exposure at the lower fluence modifies the perimeters of cells under hyposmotic stress when compared to perimeters of cells not exposed to laser in respective NaCl concentrations. Moreover, laser exposure at the higher fluence significantly (p < 0.05) modifies the perimeters of cells in 0.6% NaCl.

Figure 7 shows area values of endonuclease III deficient E. coli JW1625-1 cells exposed to low intensity laser under hyposmotic stress. Cells not exposed to laser in hyposmotic stress present a significant (p < 0.05) increase of area when compared to cells not exposed to laser in 0.9% NaCl. Cells exposed to laser at 1 J cm⁻² in the lowest NaCl concentrations (0.0 and 0.3%) present increased area when compared to cells in 0.9% NaCl and these present an area significantly increased when compared to cells not exposed to laser. Data in figure 7 show also that exposure to red laser at 8 J cm⁻² significantly (p < 0.05) alters the area of cells in the higher hyposmotic stress and in 0.9% NaCl.

Data from perimeter measurements (figure 8) confirm the data of area of E. coli JW1625-1 cells. However, perimeter of cells not exposed to laser in 0.6% NaCl is similar to perimeter of cells in 0.9% NaCl. Perimeter of cells exposed to laser (1 and 8 J cm⁻²) in 0.9% NaCl is similar to perimeter of cells not exposed to laser in the same NaCl concentration, and also there is no significant difference (p > 0.05) between perimeter of cells exposed to laser at the higher fluence in 0.0% NaCl and perimeter of cells not exposed to laser in the same condition.

4. Discussion

Our results show that exposure to low intensity red laser increases the viability of wild type E. coli (AB1157) cells under middle hyposmotic stress (figure 1). This agrees with previous data that exposure to infrared laser increases the survival of wild type E. coli cells in the stationary growth phase [7]. Also, the biostimulation effect was obtained in mammalian cell cultures under stress conditions by low serum concentration [19]. On the other hand, red laser decreases the viability of endonuclease III deficient E. coli (JW1625-1) cells (figure 2) under middle hyposmotic stress. These results reinforce that laser-induced effects are dependent on functioning DNA repair mechanisms, at least these related to endonuclease III, and physiologic or stress conditions of cells [25].

Bacterial filamentation is characterized by an anomalous growth of bacteria, in which cells continue to elongate but there is no septa formation [28]. This bacterial morphological change occurs in bacterial cultures submitted to environmental agents, both natural and man-made, which perturb the integrity of DNA [28, 29] and is part of the so-called SOS function [30]. Previous results have demonstrated that low intensity lasers induce filamentation phenotype in wild type and in E. coli cells deficient in DNA repair mechanisms [22, 25]. However, induction of this phenotype in bacterial cultures exposed to low intensity lasers under stress conditions has not been evaluated yet. Analysis of figure 3 suggests that hyposmotic stress alone induces filamention in the wild type, as well as low intensity red laser exposure in wild type E. coli cells in physiologic NaCl concentration (0.9% NaCl). It has been reported that hyposmotic stress [31] and low intensity red laser [25, 26] induces this phenotype in bacterial cultures. However, laser-induced filamentation is dependent on NaCl concentration, because filamentation is low in bacterial suspensions under hyposmotic stress, except at the higher hyposmotic stress (0.0% NaCl). In fact, laser exposure decreases filamentation phenotype in bacterial suspensions at 0.3 and 0.6% NaCl. Interestingly, for endonuclease III deficient E. coli cells, filamentation phenotype is lower in bacterial suspension under hyposmotic stress than in bacterial suspension in physiologic NaCl concentration (figure 4). In agreement with previous data [26], low intensity red laser induces bacterial filamentation in endonuclease III deficient E. coli cells, but it is not capable of altering the filamentation induction in bacterial cells under hyposmotic stress, except for bacterial cells in 0.6% NaCl. These results from wild type and endonuclease III deficient cells demonstrate for the first time that low intensity red laser exposure interferes in filamentation phenotype induction by hyposmotic stress but this effect is dependent on repair of oxidative DNA lesions, at least those related to endonuclease III.

Morphological analysis (area and perimeter measurements) was performed in E. coli cells exposed to low intensity red laser under hyposmotic stress. Area and perimeter of wild type E. coli cells are not modified by hyposmotic stress (figures 5 and 6). Laser exposure alters the morphology of cells under this condition, mainly at the lower fluence (1 J cm⁻²), but not in cells in 0.9% NaCl. These results reinforce that low intensity lasers' effects are measurable in cells under stress conditions. Differently, morphological parameters are modified in endonuclease III deficient E. coli cells under hyposmotic stress (figures 7 and 8). The different response of these cells to hyposmotic stress could be related to mechanisms involved with the filamentation phenotype because endonuclease III cultures have less bacterial filaments than wild type cultures under hyposmotic stress. Also, laser exposure at low fluence seems to decrease these morphological alterations and, at the higher fluence, laser exposure abolishes the effects of low NaCl concentrations on cells. Area and perimeter are increased in endonuclease deficient E. coli cells exposed to laser. Low intensity red laser was demonstrated to alter slow potassium currents [32] and membrane conductance through K⁺ and Ca²⁺ channels is increased after red laser exposure [33]. In addition, infrared laser (810 nm) raises mitochondrial membrane potential and reduces intracellular calcium concentrations [34]. At least in part, results from morphological analyses could be explained by alterations on ion channels in membrane E. coli cells. On the other hand, results from endonuclease III deficient E. coli cells, and those from filamentation assay, suggest that effects of low intensity red laser on cells under hyposmotic stress depend on DNA repair mechanisms involved in repair of oxidative DNA lesions.

5. Conclusions

Low intensity red laser exposure has different effects on survival, filamentation phenotype and morphology of wild type and endonuclease III deficient cells under hyposmotic stress. Thus, our results suggest that therapies based on low intensity red lasers could take into account physiologic conditions and genetic characteristics of cells.

Acknowledgment

This work was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Low intensity red laser action on Escherichia coli cultures submitted to stress conditions

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Abstract

1. Introduction