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Photodynamic
Inactivation of Antibiotic Resistant Strain of Pseudomonas Aeruginosa by
Porphyrins Induced by Delta-aminolaevulinic Acid
Sharma, Mrinalini; Bansal, Harsha; Gupta, Pradeep Kumar
Originally Published:20020901.
The
bacterial resistance to antibiotic treatment in hospitalized patients is a
growing problem. Photodynamic therapy (PDT) is a promising approach for
management of antibiotic resistant bacterial. It involves the killing of
target cells by reactive oxygen species produced by interaction of cell
bound photosensitizing compound with light of the appropriate wavelength2.
It has been shown that a number of photosensitizers are phototoxic to Gram
positive bacteria3. However, Gram negative bacteria are relatively
resistant to the photosensitizing effects of exogenous photosensitizers due
to the presence of highly organized outer membrane, which hinders the
uptake of photo sen sitizer3,4. In addition to the use of exogenous photo sensitizers,
bacteria can also be photoinactivated by enhancing their endogenous
production of porphyrins by addition of ALA, a precursor for haem
synthesise. For Gram negative bacteria the use of ALA is a better option
since unlike exogenously administered porphyrins which are less permeable,
ALA can penetrate Gram negative bacteria through the hydrophilic pores
present in the membranes.
Photodynamic
inactivation of Gram negative bacteria such as Haemophilus parainfluenze5,
Escherichia Coli B6 and Esch. coli hemA87 by porphyrins induced by ALA has
been reported. The photodynamic inactivation of P. aeruginosa with
porphyrins induced by ALA has also been attempted using photoirradiation at
630 nm8. However, the results were not very encouraging. Negligible photoinactivation
was observed in exponentially growing phase cells. This was attributed to
the synthesis of large amounts of photodynamically inactive porphyrinogens
and less accumulation of photodynamically active protoporphyrins. Further,
the use of 630 nm light for irradiation results in lower excitation of the
porphyrins due to less absorption at this wavelength. Enhanced photodynanic
inactivation of cells by porphyrins induced by ALA may be possible by
increasing the synthesis of photodynamically active protoporphyrins. It has
been reported that certain thiol compounds like GSH can enhance the
biosynthesis of porphyrins in bacteria. The effect of GSH on
photoinactivation of cells by ALA was therefore studied.
Materials
& Methods
ALA was
obtained from Sigma Chemical Co. St. Louis, USA. Stock solution (100 mM) of
ALA in phosphate buffer (PBS, pH 7.4) was prepared immediately before use.
Yeast extract, tryptone soya broth, GSH and PBS were obtained from
Hi-media, Mumbai, India.
P.
aeruginosa cells were oxidase positive and resistant to antibiotics
ampicillin, ceftazidime, gentamycin, and netillin. These were recovered
from clinical material submitted to the bacteriological laboratory,
Choithram Hospital, Indore, India.
Culture
maintenance and experiments were carried out at the Centre for Advanced
Technology, Indore, India.
Cells
were grown in growth medium containing 10 g/l tryptone soya broth and 5 g/l
of yeast extract. An aliquot of the culture was transferred to a fresh
growth medium and grown at 370C to log phase in a shaking incubator. This
culture was centrifuged (10,000 rpm, 10 min) and resuspended in fresh
growth medium. ALA (2.0 mM was added to the cell suspension which was
divided into four aliquots. These samples were incubated with ALA for 1, 2,
3 and 4 h in the dark. ALA at this concentration did not show any dark
toxicity. Subsequent to incubation with ALA in growth medium for different
durations, cells were washed by centrifugation and the cell pellet was
suspended in PBS. Porphyrins synthesized by cells were monitored using
fluorimeter (Spex Fluorolog, USA). The excitation wavelength used was 405
nm and emission was monitored from 550 to 750 nm. The cell number in the
suspension was determined by estimating the protein concentration by Lowry's
method10. All operations subsequent to addition of ALA were carried out in
the dark. To study the effect of GSH on ALA induced porphyrins, cells
suspended in PBS following ALA treatment for 4 h were incubated with
different concentrations of GSH for 15 min. This protocol was followed as
incubation of cells with ALA and GSH simultaneously for 4 h led to some
growth inhibition. The synthesis of porphyrins in the presence of GSH was
monitored by recording the fluorescence spectra as described earlier. Fluorescence
spectra of cells after irradiation was also measured to study the effect of
light on cell bound porphyrins.
For
studying photoinactivation, cells incubated with ALA for 4 h were suitably
diluted, transferred to a quartz cuvette and irradiated with continuous
stirring using 450W Xenon lamp, equipped in Spex fluorimeter. Excitation
monochromator was set at 405 nm. Fluence rate at the sample position was
12.5 mW/cm2 as measured by power meter Scien-Tech ( USA ). Cells were
exposed to light for 30, 60 and 90 min, which corresponded to light doses
of 22.5, 45.0 and 67.5 kJ/m2 respectively. The effect of GSH on
photoinactivation of cells was assessed by irradiating the ALA treated
cells in the presence of GSH. GSH (15 mM) was added 15 min prior to light
exposure. Aliquots of the irradiated cell suspension were removed at
various time intervals for survival assay. After appropriate dilutions,
cells were plated on solid growth medium. The number of colony forming
units (cfu) were counted in cells treated with ALA with and without GSH in
the dark as well as on irradiation after 24 h of incubation at 37 deg C.
Results
& Discussion
Fluorescence
emission spectrum of cells incubated with ALA for 4 h in growth medium is
shown in Fig. I. The peaks observed at 635 nm and 703 nm correspond with
protoporphyrins and peaks at 617 and 680 nm correspond to hydrophilic
porphyrins like coproporphyrins/uroporphyrins8. The intensity of 635 nm
peak was higher as compared to 617 nm. This suggests enhanced synthesis of
hydrophobic protoporphyrins. This was also confirmed by the absence of
porphyrin peaks in the cell free growth medium (data not shown).
The
synthesis of protoporphyrins as a function of time in cells incubated with ALA
in growth medium is shown in Fig. 2. The fluorescence peak intensity at 635
nm was found to increase slowly at first and then increased rapidly beyond
2 h of incubation and plateaued beyond about 4 h of incubation. This
appears to be primarily due to change in the number of cells in the growth
medium. The intensity of protoporphyrin beyond 3 h was found to be more or
less constant possibly due to saturation of the enzymes involved in haem
biosynthesis.
The
effect of GSH on ALA induced porphyrins was studied by incubating cells
treated with ALA for 4 h with different concentrations of GSH for 15 min in
PBS. Up to 5 mM concentration of GSH no change was observed in porphyrin
synthesis as compared to cells treated with ALA alone. At 10 mM there was a
slight increase in peak intensity at 635 nm but the increase was not
significant (data not shown). However, when GSH concentration was increased
to 15 mM there was significant enhancement in peak intensity at 635 nm
(Fig. 3) indicating increase in protoporphyrin synthesis.
In order
to study the photodynamic effects of porphyrins accumulated by cells, cells
incubated in ALA for 4 h were irradiated in PBS with and without GSH (15
mM) at different light doses (Fig. 4). Large decrease in cell survival was
observed in cells treated with GSH as compared to cells without GSH. Cell
death was 85 per cent as compared 10 per cent observed without GSH for the
same light dose used (22.5 kj/M2). However, no cell death was observed in
cells incubated with ALA alone or ALA treated with GSH (15 mM) in the dark.
One
reason for enhanced photoinactivation in the presence of GSH could be due
to increased synthesis of protoporphyrins. However, increased phototoxicity
observed in this study is in contrast to the antioxidant effects of GSH
reported earlier 11,12. In order to understand this, the effect of light on
cell bound protoporphyrins in the presence and absence of GSH was studied
by measuring the fluorescence spectra of cells (Fig. 5A & B).
Irradiation of cells in the absence of GSH led to decrease in the intensity
of protoporphyrin peak (635 nm) (Fig. 5A) and increase in the 617 nm peak
due to hydrophilic porphyrins. This suggests that photo irradiation alters
enzyme activities responsible for conversion of hydrophilic porphyrins to
protoporphyrinogen which would lead to blockage further down in the haem
pathway and result in accumulation of hydrophilic porphyrins13. Irradiation
in the presence of GSH showed no increase in the concentration of
hydrophilic porphyrins. Irradiation in the presence of GSH at a lower light
dose (22.5 kJ/m^sup 2^) showed an increase in the peak intensity of 635 nm
indicating an increase in the synthesis of protoporphyrins. The increase
was almost three times that of ALA treated cells incubated with GSH in the
dark. In addition, a new peak appeared at 596 nm indicating formation of
metalloporphyrins14 and prominent shoulders were also seen at 653 and 672
nm, which may be the photoproducts of protoporphyrins. However, at a higher
light dose (67.5 kJ/m^sup 2^) the protoporphyrin peak intensity reduced
indicating photobleaching. The increased protoporphyrin synthesis on
irradiation may be due to oxidation of reduced porphyrinogen. GSH is known
to interact with mitochondrially generated H202 in the presence of metals
and promote reactive oxidant species which in turn oxidize reduced
porphyrins (coproporphyrinogens and uroporphyrinogens15)is. We therefore
speculate that GSH may be interacting with the H^sub 2^O^sub 2^ generated
during photodynamic reaction and promoting the oxidation of reduced
porphyrinogens present in abundance in these cells8. Enhanced formation of
protoporphyrins and their photoproducts in the presence of GSH during
irradiation may be contributing to the observed increase in the photosensitivity
of cells.
To
conclude, our results show that the photodynamic efficacy of ALA induced
porphyrins for inactivation of antibiotic resistant strain of P. aeruginosa
can be enhanced by incubating the ALA treated cells with GSH and
irradiating at 405 nm in the presence of GSH. These findings may be useful
for inactivation of antibiotic resistant strain of P. aeruginosa causing
localized infections such as in bums and wounds in patients. Use of ALA has
an added advantage as being a natural constituent of the body it can be
administered both topically as well as systemically without toxicity.
(C) 2002 Indian Journal of Medical
Research. via ProQuest Information and Learning Company; All Rights
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