ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2005, p. 2822–2827 Vol. 49, No. 7
0066-4804/05/$08.00
0 doi:10.1128/AAC.49.7.2822–2827.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Helicobacter pylori Accumulates Photoactive Porphyrins and Is Killed
by Visible Light
Michael R. Hamblin,1,2* Jennifer Viveiros,1 Changming Yang,1,2 Atosa Ahmadi,1
Robert A. Ganz,3,4 and M. Joshua Tolkoff5
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts1; Department of Dermatology,
Harvard Medical School, Boston, Massachusetts2; Sterilite LLC, Minnetonka, Minnesota3; Department of
Gastroenterology, Abbott-Northwestern Hospital, Minneapolis, Minnesota4; and Seedling
Ventures LLC, Boston, Massachusetts5
Received 21 September 2004/Returned for modification 22 September 2004/Accepted 7 March 2005
Helicobacter pylori colonizes the mucus layer of the human stomach and duodenum, causes chronic gastritis,
gastric ulcer, and is a risk factor for gastric adenocarcinoma. There is a 20% failure rate in antibiotic therapy,
which is increasingly due to antibiotic resistance and necessitates the search for alternative antimicrobial
methods. We have discovered that H. pylori when cultured in liquid medium, accumulates significant quantities
of coproporphyrin and protoporphyrin IX, both in the cells and secreted into the medium. These photoactive
porphyrins lead to cell death (up to 5 logs) by photodynamic action upon illumination with low doses of visible
light, with blue/violet light being most efficient. The degree of killing increases with the age of the culture and
is greater than that found with Propionibacterium acnes (another bacterium known to be photosensitive due to
porphyrin accumulation). Both virulent and drug-resistant strains are killed. The data suggest that photo-
therapy might be used to treat H. pylori infection in the human stomach.
Helicobacter pylori is a gram-negative microaerophilic bacte- mycoplasma, fungi, and viruses can be killed by photodynamic
rium which selectively colonizes the mucus layer of the human therapy, and recently photodynamic therapy has been investi-
stomach and duodenum (7). As more than 50% of the world gated as a treatment for infectious disease (12). The gram-
population is infected and in some countries infection rates positive bacterium that causes acne, Propionibacterium acnes, is
approach 90%, H. pylori can be termed the world’s commonest killed by both blue and red light in the absence of any added
infectious agent (19). H. pylori infection has been shown to be photosensitizers or dyes (2, 18); recently the Food and Drug
strongly associated with the presence of inflammation and Administration approved a high-intensity narrow-band blue
chronic gastritis, and once acquired, H. pylori persists, usually light therapy for the treatment of acne vulgaris (8).
for life, unless eradicated by antimicrobial therapy (15). It is In the present report we demonstrate that all tested strains
now known that H. pylori is a major cause of peptic ulcer of H. pylori, including virulent and multiply drug-resistant
disease, and in 1994, the International Agency for Cancer strains, are killed by exposure to otherwise harmless levels of
Research declared that H. pylori was a carcinogen of humans visible light. We have identified the porphyrins responsible for
and was implicated in the development of gastric cancer (11). this photosensitivity and the optimum wavelength of light. We
The most common antibacterial treatment regimens include suggest that delivery of light into the human stomach can be
bismuth, metronidazole, tetracycline, and a proton pump in- used as an effective therapy for H. pylori infection.
hibitor, or clarithromycin in combination with a proton pump (Presented in part at the 30th Annual Meeting of the Amer-
inhibitor and amoxicillin (10). Combinations such as the above ican Society for Photobiology, Quebec City, Canada, July 2002;
lead to eradication rates of about 80% but at the expense of Digestive Disease Week 2003, Orlando, Fla., May 2003; and
side effects and possible poor patient compliance. Increasing 10th Congress of the European Society for Photobiology, Vi-
development of antibiotic resistance among H. pylori isolates enna, Austria, September 2003.)
and the existence of nonresponsive patients suggest that alter-
native strategies for H. pylori eradication be sought (22).
Photodynamic therapy uses the combination of nontoxic MATERIALS AND METHODS
dyes (frequently porphyrins or their derivatives) and harmless Bacterial strains and culture conditions. The following H. pylori strains were
visible light to produce cytotoxicity via generation of reactive used: ATCC 43504 and 49503, are laboratory-adapted strains. ATCC 700824,
oxygen species (25). Photodynamic therapy has been clinically also known as J99, is a cagA vacA virulent strain whose genome has beensequenced. Clinical isolate (CI) 1 (previously known as Leung) was provided by
approved for various malignant, premalignant, and ophthalmo- David Cave (St. Elizabeth’s Medical Center, Brighton, MA), while CI2 (42-year-
logic conditions (6). It has long been known that many micro- old female with gastritis) and CI3 (75-year-old female with gastric ulcer) are
organisms including gram-negative and gram-positive bacteria, from patient biopsies taken at Abbott-Northwestern Hospital, Minneapolis, MN.
CI4 is a clinical isolate provided by D. Y. Graham (VA Medical Center, Hous-
ton, TX) from a patient with gastritis who had failed antibiotic therapy. It
exhibited MICs for clarithromycin 32 g/ml and for metronidazole 128 g/
* Corresponding author. Mailing address: Wellman Center for Pho- ml.
tomedicine, Massachusetts General Hospital, 40 Blossom Street, Bacteria were routinely grown in liquid medium consisting of Brucella broth
BAR314B, Boston, MA 02114-2698. Phone: (617) 726-6182. Fax: (617) supplemented with 10% fetal bovine serum and an antibiotic mixture of 10 g/ml
726-8566. E-mail: hamblin@helix.mgh.harvard.edu. vancomycin, 5 g/ml trimethoprim, 6 g/ml nalidixic acid, and 5 g/ml ampho-
2822
VOL. 49, 2005 PHOTOTHERAPY FOR H. PYLORI INFECTION 2823
tericin B. The solid medium consisted of the former ingredients with 1.5% agar. RESULTS
One ml of frozen stock (medium described above plus 20% glycerol) was added
to 10 ml medium in a 25-ml Erlenmeyer flask and incubated in a microaerophilic H. pylori is killed upon illumination. Initial experiments
atmosphere (GasPak jar with Campypak generators, Fisher Scientific, Hampton, showed that H. pylori was killed by illumination with low levels
NH) and rotation at 200 rpm at 37°C. After 24 h the optical density (OD) had of visible light. As shown in Fig. 1A, a suspension obtained
generally risen to 0.5, after 48 h to 1, after 72 h to 2, and after 96 h to 
3. P. acnes
(ATCC 6919) was grown in reduced thioglycolate broth (Difco 243210) in an- from a 3-day-old culture of ATCC 49503 illuminated with
aerobic jars for 48 h when the OD was 3 and on agar plates prepared from the broad-band white light (400 nm delivered at an irradiance of
same medium. Escherichia coli (ATCC 53868) was grown aerobically in brain 100 mW/cm2) gave a linear semilogarithmic plot of survival
heart infusion broth and on agar plates prepared from the same medium. fraction versus fluence. Thirty J/cm2 gave greater than 5 logs of
In vitro light delivery to H. pylori. After incubation, 3 ml of bacterial suspen- killing (99.999%). The time for 30 J/cm2 to be delivered at
sion was removed and spun down in a centrifuge (2,000  g), and the pellet was 2
resuspended in 3 ml of sterile phosphate-buffered saline. Broad-band white light 100 mW/cm is 5 min, and control suspensions of this mi-
illumination was carried out with a Spot Light Source with adjustable power croaerophilic organism exposed to the laboratory air in the
output (model L2859-07, Hamamatsu Photonics KK, Bridgewater, NJ) fitted dark did not suffer any significant loss of viability. Indeed in
with a 400-nm long-pass filter and adjusted to give a 2-cm-diameter spot with an later work we exposed H. pylori suspensions to room air for up
irradiance of 100 mW/cm2 measured with a power meter (model K1700, Inter- to 1 hour without loss of viability (data not shown). As a
national Light Inc, Newburyport, MA). Fifty-nm interference band pass filters
(Omega Optical Inc, Brattleboro, VT) were used in conjunction with the Spot control for a nonphotosensitive bacterium, we used E. coli and
Light Source to deliver light confined to the following wavelength ranges: 375 to illuminated it under the same conditions as H. pylori. There
425, 425 to 475, 475 to 525, 525 to 575, 575 to 625, and 625 to 675 nm with spot was no loss of viability of E. coli after 30 J/cm2 of white light
sizes 2 cm in diameter and irradiances of 100 mW/cm2 as measured with the had been delivered, suggesting that H. pylori is at least 100,000
power meter.
A diode laser capable of emitting 400 mW total power at 405 5 nm was times more photosensitive than E. coli.
obtained from Nichia Chemical Industries Corp. (Mountville, PA). It consisted Violet/blue light is most effective. To determine which wave-
of 20 individual GaN laser diodes coupled into 20 individual fibers and brought lengths of the visible range between 400 and 700 nm were most
together to form a 650-m coupling diameter, numerical aperture of 0.20, at the effective in killing H. pylori we used six 50-nm band pass filters
laser output port (SMA Connector). It provided a 2-cm-diameter spot with an centered on wavelengths of 400, 450, 500, 550, 600, and 650 nm
irradiance of 100 mW/cm2 to illuminate H. pylori. In some experiments E. coli
was illuminated in the same manner as a control nonphotosensitive organism. to isolate specific colors of light. As shown in Fig. 1B the range
Two hundred l of bacterial suspension was added to each well of a hanging drop between 375 and 425 nm (blue/violet light) was clearly the most
slide (Fisher Scientific) that was placed on a black background to avoid reflec- effective followed by the range of 425 to 475 nm (blue), with
tance of light. The temperature of the suspension did not rise during the illu- the other four ranges producing only a slight killing effect.
mination. All tested strains are photosensitive. We therefore obtained
Survival fraction determination. After each predefined fluence of light had
been delivered to one well, a 20-l aliquot of suspension was removed and 10 l a diode laser that delivered 405 nm light (2 nm) at a total
of this was subjected to 5 serial tenfold dilutions in PBS. The original 10 l and power of 160 mW out of the fiber. This was used to illuminated
10 l of each dilution were streaked horizontally on square agar plates according bacterial suspensions and to test generality of this observation
to the method of Jett et al. (16). The maximum time between illumination and using more strains of H. pylori as shown in Fig. 1C. All tested
plating was 10 minutes. These plates were incubated at 37°C in stationary mi- strains were killed at least 99.9% by 20 J/cm2 of 405-nm light.
croaerophilic jars for 4 days (for H. pylori) or anaerobic jars for 2 days (P. acnes)
until countable colonies appeared. Surviving CFU were counted and recorded However there were differences between strains. It appears
for analysis. Survival fractions were determined relative to unilluminated bacte- that the laboratory-adapted strains (ATCC 49503 and 43504)
rial suspensions that had been exposed to laboratory air in the same wells for the are more sensitive (1 to 2 logs) than the more recent clinical
same time as the illumination. isolates. The fact that strain J99 that expresses virulence fac-
Fluorescence spectroscopy. The culture supernatant (2 ml) from suspensions
of H. pylori that would subsequently be illuminated was added to 1 ml of a tors and a doubly antibiotic resistant strain (CI4) were both
mixture of 0.1 M NaOH/1% sodium dodecyl sulfate (SDS) and allowed to stand killed suggests that phototherapy might be applied to treat
in the dark for 1 day. Fluorescence was measured on a fluorimeter (Fluoromax antibiotic-resistant disease.
3, SPEX Industries, Edison, NJ), with excitation at 405 nm and emission scanned Photosensitivity depends on culture age and is greater in H.
from 580 to 720 nm. Peak heights were correlated with the reciprocal of the pylori than P. acnes. We observed that the extent of killing of
survival fraction after 10 J/cm2 405-nm light had been delivered.
Identification of porphyrins. H. pylori culture medium supernatant (4 ml) was H. pylori bacterial suspensions observed by delivering defined
added to 1 ml of 0.1 M NaOH and 1 ml of chloroform. The aqueous layer was fluences depended on the age of the liquid culture providing
extracted with a mixture of ethyl acetate and glacial acetic acid (10 ml, 8:1) and the suspension. We also wished to compare the relative pho-
the organic layer washed with 1 ml of 0.1 M sodium bicarbonate and then tosensitivity of the previously best-known porphyrin accumu-
deionized water and evaporated. The residue was dissolved in 200-l capillary lating bacterium, P. acnes. Figure 2A compares the killing
electrophoresis (CE) buffer (10 mM 2-[N-cyclohexylamino]ethanesulfonic acid
and 75 mM sodium dodecyl sulfate at pH 10) for CE-laser-induced fluorescence curves of 1-, 2-, and 4-day-old cultures of H. pylori and P. acnes.
(CE-LIF) analysis. The weighed dry pellet of H. pylori or P. acnes (ca. 20 mg) was As can be seen there is a very large difference between the
added to 4 ml of 0.1 M NaOH and digested in the dark at room temperature for photosensitivity of the two bacteria. P. acnes requires two to
48 h. The mixture was extracted as described for the supernatant. three times the light to produce killing 100 to 1,000 times less
Coproporphyrin I dichloride (CP, FW 727.6) and protoporphyrin IX (PPIX, than that seen with H. pylori. The degree of photosensitivity of
FW 562.7) were from Sigma. PPIX was dissolved in CE buffer to give 1.2  104
M solution and CP in 0.1 N NaOH to give a 1.6  103 M solution. Calibration H. pylori increases with the age of the culture with a 4 day old
curves were constructed with dilutions from 2.5  108 to 64  108 M. CE-LIF culture being most sensitive. Cultures significantly older than 4
analysis used a Beckman P/ACE MDQ system (Beckman Coulter, Fullerton, days run the risk of becoming coccoid and nonculturable (5,
CA, USA) and a fused silica capillary (60 cm by 150 m internal diameter) with 27). There is some variability (1 to 2 logs) in the light-induced
a detection window at 50 cm from the inlet and a 488-nm argon laser for
excitation. Separation voltage was 
30 kV in CE buffer. The recorded peak areas killing of H. pylori cultures even of the same strain and chro-
were used in plotting the standard curves and determining sample concentrations nological age. This is due to the variation in the amounts of
of CP and PPIX. porphyrins (1- to 2-fold) produced by these cultures. The fac-
2824 HAMBLIN ET AL. ANTIMICROB. AGENTS CHEMOTHER.
tors that influence the quantitative differences in porphyrin
accumulation in H. pylori cultures of the same strain and chro-
nological age are at present unknown.
H. pylori produces porphyrin fluorescence that correlates
with phototoxicity. The data suggested that the reason H. pylori
was killed by visible light with a peak in the action spectrum
about 400 nm was due to the bacteria accumulating a metal-
free porphyrin that produces reactive oxygen species upon
illumination. This hypothesis was tested by carrying out fluo-
rescence emission spectroscopy on the 4-day-old H. pylori pel-
let that had been dissolved in NaOH/SDS. We excited the
solution at 405 nm and scanned the emission between 580 and
720 nm.
Figure 2B shows that the supernatant contains an emission
peak centered at 622 nm almost midway between the emission
peaks of CP (610 nm) and PPIX (632 nm). We correlated the
measures of the fluorescence intensity in the culture superna-
tants with the amount of killing observed in many experiments
that gave variable degrees of light-mediated killing and were
carried out with different strains of H. pylori and particularly
with cultures of different ages. The reciprocal of the survival
fraction after 10 J/cm2 blue light was taken as the measure of
cytotoxicity. There was an excellent positive correlation over
several orders of magnitude between the porphyrin fluores-
cence in the medium and the cytotoxicity with an R-value of
0.878 (P  0.001) as shown in Fig. 2C. The variation in por-
phyrin content and photosensitivity was largely due to using
cultures that had been grown for various lengths of time (1, 2,
3, and 4 days).
H. pylori produces both CP and PPIX. To further identify
and confirm the existence of free porphyrins in the four-day
old culture supernatant and bacterial pellets, we carried out
analysis by solvent extraction and CE-LIF. Representative
electropherograms of the culture supernatant from ATCC
49503 and a mixture prepared from authentic CP and PPIX are
shown in Fig. 3. We were able to prepare a calibration curve
from various concentrations of CP and PPIX standards that
allowed us to quantitatively compare the concentrations of the
porphyrins in supernatants from both the H. pylori strains and
from P. acnes.
We were also able to measure concentrations of porphyrins
extracted from bacterial cell pellets but due to the sensitivity of
the laser-induced fluorescence detector we needed to grow
enough bacteria to make a pellet weighing about 15 mg dry
weight (from approximately 20 ml of 4-day-old suspension). As
the pellet isolated from P. acnes was much larger than that
isolated from H. pylori we determined the dry weight after
freeze drying before dissolving the pellet in NaOH/SDS. The
results are presented in Table 1. The concentrations of both
CP and PPIX measured in the supernatants from the five
different H. pylori strains and from P. acnes were roughly com-
parable, ranging from 10 to 20 nM for CP and from 12 to 42
nM for PPIX. In all cases the PPIX concentration was larger
than that of CP (between 1.2 and 2.7 times higher).
FIG. 1. A) Fluence-dependent killing of H. pylori ATCC 49503
after exposure to broad-band white light, 400 nm. E. coli is unharmed
after similar illumination. Values are means of three independent
experiments and bars are standard errors. B) Fluence-dependent kill-
ing of H. pylori ATCC 49503 after exposure to light produced by 50-nm seven strains of H. pylori illuminated with light from a 405-nm diode
band-pass filters. Values are means of three independent experiments laser. Values are means of three independent experiments and bars are
and bars are standard errors. C) Comparison of photosensitivity of standard errors.
VOL. 49, 2005 PHOTOTHERAPY FOR H. PYLORI INFECTION 2825
DISCUSSION
We have shown for the first time that H. pylori, when grown
in liquid medium, naturally accumulates sufficient photoactive
porphyrins to allow it to be efficiently killed after illumination
with low fluences of blue light. This finding suggests that a
novel form of phototherapy could be applied in the human
stomach to eradicate the infection. There have been scattered
reports of different species of bacteria exhibiting photosensi-
tivity due to the accumulation of porphyrins. Many of these
concern the gram-positive anaerobe P. acnes that is the cause
of the human skin disease acne. P. acnes has been reported to
be killed by both blue and red light (2, 18), and recently the
Food and Drug Administration approved a high-intensity nar-
row-band blue light therapy for the treatment of acne vulgaris
(8). However, our data comparing the photosensitivity of H.
pylori and P. acnes demonstrate that, although they accumulate
comparable amounts of porphyrins, the former is several thou-
sandfold more photosensitive.
Our finding of the relative resistance of P. acnes to blue light
killing is in agreement with a report from Ashkenazi et al. (2),
who needed to use repetitive illumination of P. acnes in order
to achieve satisfactory levels of killing. They achieved less than
2 logs of killing after 75 J/cm2 blue light, but this increased to
4 logs of killing after two similar illuminations 24 h apart. The
explanation for the observation that P. acnes and H. pylori
make similar amounts of porphyrins but have very different
responses to illumination is at present unclear. There are
clearly important differences between H. pylori and P. acnes,
such as Gram stain status (H. pylori is gram-negative while P.
acnes is gram-positive) and H. pylori is microaerophilic while P.
acnes is anaerobic, but there are so few examples of natural
porphyrin-producing bacteria that the determinants of relative
susceptibilities to photoinactivation remain unknown.
It has long been known that some bacteria accumulate por-
phyrins under some circumstances, and consequently exhibit a
red fluorescence under UVA or blue light illumination and are
susceptible to killing upon illumination (18). This was reported
to be common among anaerobic species (4) and red-fluores-
cent pus was reported to be present in infections (3). The
phenomenon has been noted in a group of anaerobic species
that are mainly oral pathogens and were previously known as
black-pigmented Bacteroides species but have now been reclas-
sified as Porphyromonas and Prevotella species (17). These bac-
teria depend largely on external heme (either hemoglobin or
hemopexin) to satisfy their demand for iron (20) and to a
greater or lesser extent accumulate a black pigment intracel-
lularly that consists of an iron-containing heme aggregate (e.g.,
hematin) together with various amounts of iron-free PPIX (28,
29). If they accumulate PPIX they will be photosensitive (13),
however, if they mainly have hematin they will not be photo-
FIG. 2. A) Comparison of photoinactivation using 405-nm laser
light of H. pylori ATCC 49503 and P. acnes grown in liquid medium for
various lengths of time. Values are means of three independent ex-
periments and bars are standard errors. B) Fluorescence spectroscopy
of 4-day-old culture medium from ATCC 49503 compared to CP and
PPIX standards. C) Correlation between height of fluorescence emis-
sion from culture supernatants and cytotoxicity expressed as reciprocal
of surviving fraction after 10 J/cm2 of 405-nm light.
2826 HAMBLIN ET AL. ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. A) Capillary electrophoresis of standard mixture of CP (peak 1) and PPIX (peak 2). B) Capillary electrophoresis of extract from culture
supernatant of 4-day-old H. pylori ATCC 49503. Peaks marked * may be artifacts or very low concentrations of porphyrins with more than four
carboxyl groups.
sensitive, as iron-containing tetrapyrroles do not carry out the evolutionary advantage to H. pylori accumulating these com-
appropriate photochemistry. Henry et al. showed that these pounds.
species were more sensitive when grown on media containing The finding that H. pylori porphyrin levels and susceptibility
hemin as an iron source compared to hemoglobin (14). to photokilling increases with increasing age of the culture is
A second group of porphyrin-accumulating bacteria com- also in agreement with a report of a similar finding concerning
prises species such as P. acnes and now H. pylori. Here the P. acnes (23). It is possible that the porphyrin accumulation is
porphyrins consist of mixtures of PPIX with CP and sometimes controlled by quorum-sensing mechanisms whereby bacteria
uroporphyrin (26). The vast majority of bacterial species use secrete small molecules termed autoinducers that are detected
the heme biosynthetic pathway by which (in a similar fashion to by neighboring cells and induce expression of a set of genes
mammalian cells) porphyrins are produced from the precursor involving survival protection and increased virulence (24, 31).
5-aminolevulanic acid by a well-conserved set of enzymes. The H. pylori, while not possessing autoinducer system one involv-
finding that both H. pylori and P. acnes accumulate a tetracar- ing acylhomoserine lactones, does possess the luxS gene that
boxylic porphyrin (CP) as well as PPIX shows that the porphy- codes for the biosynthesis of autoinducer AI2 (9).
rins must arise from endogenous heme biosynthesis rather By analogy with P. acnes (1), the mechanism of killing of H.
than accumulation of the porphyrins from exogenous heme, as pylori is likely to involve photodynamic action whereby the
there is no known biochemical mechanism for producing a porphyrins absorb light and produce, via the excited singlet
tetracarboxylic porphyrin from a dicarboxylic porphyrin state, a long-lived triplet state that can interact with molecular
present in heme. Normally the heme biosynthetic cycle is sub- oxygen to produce the cytotoxic species singlet oxygen. This
ject to tight feedback control to avoid producing photoactive mechanism implies that the killing will be oxygen dependent,
free porphyrins, and it is presently unclear if there is any and consideration must be given to whether there will be suf-
VOL. 49, 2005 PHOTOTHERAPY FOR H. PYLORI INFECTION 2827
TABLE 1. Strains useda Propionibacterium acnes by its endogenic porphyrins after illumination with
high intensity blue light. FEMS Immunol. Med. Microbiol. 35:17–24.
Strain Coproporphyrin (M) Protoporphyrin (M) 3. Brazier, J. S. 1990. Analysis of the porphyrin content of fluorescent pus by
absorption spectrophotometry and high performance liquid chromatogra-
H. pylori ATCC 43904 1.57  108 4.27  108 phy. J. Med. Microbiol. 33:29–34.
H. pylori ATCC 700824 1.97  108 3.82  108 4. Brazier, J. S. 1986. A note on ultra-violet red fluorescence of anaerobic
H. pylori ATCC 49503 1.53  108 3.44  108 bacteria in vitro. J. Appl. Bacteriol. 60:121–126.
H. pylori CI3 1.59  108 2.7  108 5. Cellini, L., I. Robuffo, E. Di Campli, S. Di Bartolomeo, T. Taraborelli, and
H. pylori CI1 1.02  108 1.23  108 B. Dainelli. 1998. Recovery of Helicobacter pylori ATCC43504 from a viable
P. acnes supernatant 1.37  108 3.07  108 but not culturable state: regrowth or resuscitation? APMIS 106:571–579.
8 8 6. Dougherty, T. J. 2002. An update on photodynamic therapy applications.P. acnes pellet (16.5 mg) 0.68  10 1.12  10 J. Clin. Laser Med. Surg. 20:3–7.
(1.08 ppm wt/wt) (1.53 ppm wt/wt) 7. Dunn, B. E., H. Cohen, and M. J. Blaser. 1997. Helicobacter pylori. Clin.
H. pylori pellet (15.2 mg) 0.56  108 0.88  108 Microbiol. Rev. 10:720–741.
(0.96 ppm wt/wt) (1.3 ppm wt/wt) 8. Elman, M., M. Slatkine, and Y. Harth. 2003. The effective treatment of acne
vulgaris by a high-intensity, narrow band 405–420 nm light source. J. Cosmet.
a Concentrations of CP and PPIX in H. pylori and P. acne supernatants and Laser Ther. 5:111–117.
pellets were determined by solvent extraction, CE-LIF separation, and compar- 9. Forsyth, M. H., and T. L. Cover. 2000. Intercellular communication in Hel-
ison with calibration curves. icobacter pylori: luxS is essential for the production of an extracellular sig-
naling molecule. Infect. Immun. 68:3193–3199.
10. Gene, E., X. Calvet, R. Azagra, and J. P. Gisbert. 2003. Triple vs. quadruple
therapy for treating Helicobacter pylori infection: a meta-analysis. Aliment.
ficient oxygen in the stomach mucosa to allow complete killing. Pharmacol. Ther. 17:1137–1143.
There is known to be enough oxygen present in the gastric 11. Graham, D. Y., M. F. Go, and R. M. Genta. 1995. Helicobacter pylori, duodenal
mucosa for phagocytic cells to generate reactive oxygen species ulcer, gastric cancer: tunnel vision or blinders? Ann. Med. 27:589–594.
12. Hamblin, M. R., and T. Hasan. 2004. Photodynamic therapy: a new antimi-
in a respiratory burst. In addition it is presently uncertain crobial approach to infectious disease? Photochem. Photobiol. Sci. 3:436–
whether light penetration into the lining of the human stomach 450.
will be a barrier to photoeradication. The gastric glands and 13. Henry, C. A., B. Dyer, M. Wagner, M. Judy, and J. L. Matthews. 1996.Phototoxicity of argon laser irradiation on biofilms of Porphyromonas and
crypts will be situated deep within the mucosa, and tissue is an Prevotella species. J. Photochem. Photobiol. B 34:123–128.
effective absorber and scatterer of blue light. The surface area 14. Henry, C. A., M. Judy, B. Dyer, M. Wagner, and J. L. Matthews. 1995.
of the stomach is approximately 800 cm2 and if illumination is Sensitivity of Porphyromonas and Prevotella species in liquid media to argonlaser. Photochem. Photobiol. 61:410–413.
to be carried out in a reasonable time during endoscopy it may 15. Hunt, R. H. 1996. The role of Helicobacter pylori in pathogenesis: the
be necessary to deliver optical powers as high as 50 to 100 W spectrum of clinical outcomes. Scand. J. Gastroenterol. Suppl. 220:3–9.
16. Jett, B. D., K. L. Hatter, M. M. Huycke, and M. S. Gilmore. 1997. Simplified
into the stomach. agar plate method for quantifying viable bacteria. BioTechniques 23:648–650.
It has been proposed to treat H. pylori by administering oral 17. Jousimies-Somer, H., and P. Summanen. 2002. Recent taxonomic changes
5-aminolevulanic acid that acts as a metabolic precursor of and terminology update of clinically significant anaerobic gram-negativebacteria (excluding spirochetes). Clin. Infect. Dis. 35:S17–21.
PPIX followed by illumination of the stomach lining by blue 18. Konig, K., M. Teschke, B. Sigusch, E. Glockmann, S. Eick, and W. Pfister.
light (30). However, it is well known that 5-aminolevulanic acid 2000. Red light kills bacteria via photodynamic action. Cell. Mol. Biol.
can produce PPIX in human mucosal linings (21) and the use (Noisy-le-Grand) 46:1297–1303.19. Lacy, B. E., and J. Rosemore. Helicobacter pylori: ulcers and more: the
of this drug may increase the chances of damaging the stomach beginning of an era. J. Nutr. 131:2789S–2793S, 2001.
lining. 20. Leung, K. P., P. S. Subramaniam, M. Okamoto, H. Fukushima, and C. H.
Lai. 1998. The binding and utilization of hemoglobin by Prevotella interme-
In conclusion, we have shown that all tested strains of H. dia. FEMS Microbiol. Lett. 162:227–233.
pylori naturally accumulate a mixture of PPIX and CP that can 21. Loh, C. S., D. Vernon, A. J. MacRobert, J. Bedwell, S. G. Bown, and S. B.
sensitize the bacteria to killing by visible light, particularly blue Brown. 1993. Endogenous porphyrin distribution induced by 5-aminolaevu-linic acid in the tissue layers of the gastrointestinal tract. J. Photochem.
light. This finding suggests that a novel phototherapy approach Photobiol. B 20:47–54.
may be applied in the human stomach to eliminate H. pylori 22. Megraud, F., and H. Lamouliatte. 2003. Review article: the treatment of
infection. refractory Helicobacter pylori infection. Aliment. Pharmacol. Ther. 17:1333–1343.
23. Melo, T. B., and G. Reisaeter. 1986. Photodestruction of endogenous por-
ACKNOWLEDGMENTS phyrins in relation to cellular inactivation of Propionibacterium acnes. Z.
Naturforsch. 41:867–872.
This work was supported by grants from LumeRx Inc, Seedling 24. Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu.
Enterprises LLC, and Public Health Service grant R01AI050879 to Rev. Microbiol. 55:165–199.
M.R.H. from the National Institute for Allergy and Infectious Disease. 25. Ochsner, M. 1997. Photophysical and photobiological processes in the pho-
We thank Touqir Zahra and Aamir Ahmad for technical assistance todynamic therapy of tumours. J. Photochem. Photobiol. B 39:1–18.
and Philip Levin and Robert Arcangeli for helpful discussions. We are 26. Romiti, R., M. Schaller, K. Jacob, and G. Plewig. 2000. High-performance
grateful to David Cave, David Y. Graham, and Anne Kane for gen- liquid chromatography analysis of porphyrins in Propionibacterium acnes.Arch. Dermatol. Res. 292:320–322.
erous gifts of strains. 27. Saito, N., K. Konishi, F. Sato, M. Kato, H. Takeda, T. Sugiyama, and M.
The authors have commercial associations that might pose a conflict Asaka. 2003. Plural transformation-processes from spiral to coccoid Helico-
of interest. Michael R. Hamblin is a consultant to LumeRx and Seed- bacter pylori and its viability. J. Infect. 46:49–55.
ling and received research support from LumeRx and Seedling. Robert 28. Shah, H. N., R. Bonnett, B. Mateen, and R. A. Williams. 1979. The porphyrin
A. Ganz is a stockholder in LumeRx and is a member of their scientific pigmentation of subspecies of Bacteroides melaninogenicus. Biochem. J.
advisory board and an employee of Sterilite. Sterilite received research 180:45–50.
support from LumeRx and Seedling. M. Joshua Tolkoff is a stock- 29. Shah, H. N., and S. E. Gharbia. 1993. Biochemical and chemical analyses of
holder in LumeRx and Seedling and is an employee of Seedling. black-pigmented gram-negative anaerobes. FEMS Immunol. Med. Micro-biol. 6:89–96.
30. Wilder-Smith, C. H., P. Wilder-Smith, P. Grosjean, H. van den Bergh, A.
REFERENCES Woodtli, P. Monnier, G. Dorta, F. Meister, and G. Wagnieres. 2002. Photo-
1. Arakane, K., A. Ryu, C. Hayashi, T. Masunaga, K. Shinmoto, S. Mashiko, T. eradication of Helicobacter pylori using 5-aminolevulinic acid: preliminary
Nagano, and M. Hirobe. 1996. Singlet oxygen (1 delta g) generation from human studies. Lasers Surg. Med. 31:18–22.
coproporphyrin in Propionibacterium acnes on irradiation. Biochem. Bio- 31. Winzer, K., and P. Williams. 2001. Quorum sensing and the regulation of
phys. Res. Commun. 223:578–582. virulence gene expression in pathogenic bacteria. Int. J. Med. Microbiol.
2. Ashkenazi, H., Z. Malik, Y. Harth, and Y. Nitzan. 2003. Eradication of 291:131–143.