The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Summary

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

Abstract

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens^1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region^4,6,7. There are also reports of biocidal effect of red and near infra red⁸ as well as green light⁹.

In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems¹⁰.

The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.

The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

Protocol

1. Bacterial Culture

1. Gram-negative P. aeruginosa strain PAO1 were grown in Luria Broth (LB) at 37 °C for 18 hr.
2. The culture of cells was then centrifuged at 7,500 rpm (rounds per minute) for 5 min and supernatant was removed.
3. The bacteria were resuspended in 10% LB and re-grown for another 2 hr to allow the culture to reenter stationary phase.
4. The bacteria suspension was then divided into two groups: in the first group (2 tubes) no antibiotic were added, in the second group we added the gentamycin antibiotic (50 μg/ml).

2. Determination of Colony-forming Units (CFU)

1. To determine cell viability 20 μl samples were taken from the experiment approximately every 2 hr within the time frame of 24 hr. Serial dilution of the samples were made and plated on LB agar plates and incubated overnight at 37 °C.
2. For each treatment, the CFUs per plate was determined and a comparison was made between the time periods and various treatments. The log reduction in CFU was calculated as described in Eq. (1):
   Log reduction= LogU-LogC[CFU/ml]
   Where U is the colony forming units value at each time point; CFU is the colony-forming unit while the units of CFU/ml equal to:
   CFU/ml = ( number of colonies x dilution factor )/( volume inoculated )
   And C is the CFU found in the control sample at start time. Note that U designates the colony forming factor at measurement instant.
3. The dilution factor is the number of dilutions while in each one of them the concentration of the bacteria was reduced by a factor of 10. The inoculated volume was always 200 micro liters and it is related to the size of our test tube.

Therefore to summarize the concentration point of view, the gentamycin antibiotic was at concentration of 50 μg/ml. Regarding the bacteria, until the end of the process we had overall 8 dilutions. Each dilution was by a factor of 10 and it was done in tubes of 200 μl. The starting point was 20 μl of samples added into the 200 μl tube (and thus the initial concentration was 20/200C₀=0.1C0 with C₀ being the initial concentration in the 20 μl of samples) and the final concentration was reduced by 8 orders of magnitude due to the 8 dilutions.

3. Illumination

1. CW Nd:YAG laser (wavelength of 532 nm and average optical power of 200 mW) was split into two optical paths using optical 50%/50% beam splitter. The beam diameter was about 10 mm. The exposure duration was 24 hr.
2. Q-switched pulsed Nd:YAG laser (wavelength of 532 nm, average power of 300 mW and optical peak power of 2.5 MW) was also split into two paths using optical 50%/50% beam splitter. The spot diameter was 6 mm. The pulse width of the Q-switched laser was 6 nsec and the repetition rate was 15Hz. The average power density was 106 mW/cm² and the peak power density was 8.83 kW/mm². The exposure duration was 24 hr.

Note that the bacterial suspension stirred during irradiation and it was kept under appropriate culture conditions for bacterial growth (in all tubes there was Luria Broth medium to allow the bacteria to grow).

Results

The laser based setup is schematically presented in Figure 1. The first experimental condition utilized a CW Nd:YAG laser having wavelength of 532 nm (the second harmonic of the Nd:YAG) and average optical power of 200 mW. This beam was split into two optical paths using optical 50%/50% beam splitter such that each split beam had power of 100 mW. The beam diameter was about 10 mm and thus the power density was about 100 mW/cm². The exposure duration was 24 hr. Although the illumination power i...

Discussion

Phototherapy has been a field of advanced multidisciplinary research in recent years emerging as a promising approach for treatment numerous diseases. In this context the use of light in the visible range has been extensively studied. For example, it has been found that infected wounds can be healed more effectively by exposing them to intense visible light for sterilization purposes. The mechanism of action for this approach was proven to be through the induction of light-induced oxygen radicals (ROS) which kill the bac...

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
Lauria Broth	Difco	241420
Gentamycin	Sigma	G1914
Bacto Agar	Difco	231710