CURRENTMICROBIOLOOYVol. 29 (1994), pp.

125-131
Current
Microbiology
An International Journal
9 Springer-Verlag New York Inc. 1994

Inactivation of Anaerobic Bacteria by Various Photosensitized

Porphyrins or by Hemin
Yeshayahu Nitzan, 1 Hannah M. Wexler, 3,4 Sydney M. Finegold 2,4,5
1Health Science Research Center, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
2Medical Services, Veterans Administration Medical Center, West Los Angeles, Wadsworth Division, Los Angeles, CA, USA
3Research Services, Veterans Administration Medical Center, West Los Angeles, Wadsworth Division, Los Angeles, CA, USA
4Department of Medicine, UCLA School of Medicine, Los Angeles, CA, USA
5Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA, USA

Abstract. The photodynamic effects of deuteroporphyrin (DP), hematoporphyrin derivative (HPD),

hematoporphyrin (HP), or protoporphyrin (PP) on a variety of anaerobic microorganisms were
examined in this study. The majority of the species, among the 350 strains tested, were inhibited by
concentrations of < 2.5 ~g/ml of light-activated DP. Species found to be resistant to this treatment
included Bilophila wadsworthia, Fusobacterium mortiferum, Fusobacterium varium, and Bacteroides
gracilis. These species were inhibited by concentrations of > 60 p,g/ml of DP. The porphyrin-
producing species, Porphyromonas and Prevotella spp, were all inhibited by < 2.5 txg/ml DP and
light. Comparing the photodynamic activity of the porphyrins used on Porphyromonas strains
resulted in the following pattern: DP > HPD > HP > PP. Porphyromonas spp., Gram-positive
cocci, and many Gram-positive rods (excluding clostridia) were inactivated by hemin (a metal-
containing porphyrin) at 10-20 p.g/ml. Hemin inhibitory action was not affected by light. Binding
and insertion of DP into bacteria (both inactivated and non-inactivated strains by DP and light)
were monitored by the characteristic fluorescence band of bound DP at 622 nm. Porphyromonas
spp. bound DP tightly, whereas only low binding was seen with B. wadsworthia and other
DP-resistant species. High binding of DP to B. wadsworthia can be achieved by pretreatment of the
bacteria with imipenem or cefoxitin, 13-1actam agents known to interfere with the integrity of the
cell wall. If cell wall integrity is disturbed (e.g., by these agents), inactivation of B. wadsworthia by
DP can occur.

Metal-free porphyrins have been used as anticancer
therapeutic agents in clinical medicine. When acti-
vated by light, they act selectively against solid tumors
with minimal damage to the host [5, 12]. Research in
the field of photodynamic therapy has resulted in the
development of a photodynamic modality against
microorganisms. Porphyrin-induced photodynamic ef-
fect leads to efficient antimicrobial killing upon illumi-
nation of Gram-positive bacteria, mycoplasma and
yeasts, but not Gram-negative ceils [2, 3, 10, 13, 22].
Gram-negative bacteria were found recently [26] to
be inhibited by photosensitization of porphyrins only
in the presence of polymyxin nonapeptide (PMNP).
Polymyxin nonapeptide is a non-toxic agent that
disorganizes the bacterial membrane structure [11,
29]. This disorganization of the membrane structure
allows the porphyrin to bind to the cytoplasmic
Correspondence to: Y. Nitzan
membrane of Gram-negative bacteria. The prerequi-
site for photosensitization of a microbial cell is the
binding of the porphyrin to the cytoplasmic mem-
brane. This binding is strongly pH dependent [6]. The
immediate inhibition of cell growth is accompanied
by alterations in protein, DNA, and RNA synthesis as
well as perturbed cell wall and cell membrane synthe-
sis [16, 19, 20].
Furthermore, hemin, an iron-containing porphy-
rin synthesized by most bacterial species, has shown
an antibacterial effect on Staphylococcus aureus and
other Gram-positive species [15, 21]. This antibacte-
rial activity is independent of illumination. It was also
shown that a combination of deuteroporphyrin and
hemin has a stronger effect than that of the separate
constituents and is equally efficient with or without
illumination [15].
Almost no information is available on the photo-
sensitization of anaerobic bacteria by porphyrins and
126
light. On the other hand, hemin is an important
supplement for anaerobic bacterial growth [27]. Since
photosensitization of bacterial cells is independent of
the antibiotic resistance spectrum of the treated
pathogen, the antibacterial action of porphyrins may
ultimately have therapeutic potential.
In the present work, we studied the photody-
namic effect of porphyrins on both Gram-positive and
Gram-negative anaerobic bacteria. The connection
between porphyrin binding and killing was evaluated.
In addition, the antimicrobial effect of hemin (in the
dark), as well as the effects of the combination of
deuteroporphyrin and hemin in the dark, was investi-
gated.
Materials and Methods
Bacterial cultures. All 350 strains of anaerobic bacteria tested were
isolated in the Clinical Anaerobic Bacteriology Laboratory at the
Veterans Administration Medical Center, West Los Angeles (Wad-
sworth) and were identified according to standard methods [8, 27].
Reference strains ofB. fragilis (ATCC 25285) and B. thetaiotaomi-
cron (ATCC 29741) were used as controls in the antimicrobial
susceptibility tests. These strains were tested in at least 12 different
experiments and gave consistently the same results (MICs of < 2.5
Ixg/ml and 5 ixg/ml for DT, respectively; MIC for hemin by both
strains was > 60 txg/ml). The ATCC strains were obtained from the
American Type Culture Collection (Rockville, Maryland). Strains
were subcultured on Brucella blood agar before testing, and
colonies were suspended in Brucella broth (BBL Microbiology
Systems, Becton Dickinson & Co., Cockeysville, Maryland) to
obtain 0.5 McFarland units of turbidity.
Antimierobial susceptibility tests and photosensitization proce-
dure. Minimal inhibitory concentrations (MICs) of the various
porphyrins were determined by the NCCLS-approved, Wadsworth
agar plate dilution technique outlined in NCCLS document Mll-A2
[18] and the Wadsworth Anaerobic Bacteriology Manual [27].
Briefly, Brucella agar (BBL Microbiology Systems) plates contain-
ing serial twofold dilutions of porphyrins were inoculated in an
anaerobic chamber (Anaerobe Systems, San Jose, California) with
105 CFU per spot with a Steers replicator. After 48 h of incubation
in anaerobic conditions at 37~ MICs were read as the lowest
concentration of the porphyrin permitting no growth. In order to
maintain photosensitization, the plates were placed with their
inoculated surface upwards and illuminated for 2 h in an anaerobic
chamber, with two 60-W tungsten lamps placed 30 cm above the
plates. Plates tested in the dark were transferred immediately after
inoculation into a dark container and incubated for 48 h as above.
For testing the protection of sulfhydryl reagents on hemin action,
2-mercaptoethanol or L-cysteine (final concentrations, 10 -1 to 10 -5
M) were added to the bacteria prior to hemin treatment. Brucella
agar plates used were all supplemented with 1 jxg/ml vitamin K1.
During tests with the B. wadsworthia strains, the medium was
supplemented with 1% pyruvic acid (Sigma, St. Louis, Missouri). B.
gracilis strains were tested on Brucella agar supplemented with
vitamin K1 and 0.3% formate-fumarate (final concentration).
Preparation of porphyrin solutions. Porphyrin stock solutions were
prepared by dissolving 5 mg of the porphyrin (powder) in 0.2 ml 1 N
NaOH; 1.8 ml of 0.85% NaC1 solution was then added, and the
mixture was vortexed. Stock solutions were kept in the dark at
CURRENTMICROBIOLOGYVo1.29 (1994)
+4~ Deuteroporphyrin IX dihydrochloride was obtained from
Porphyrin Products (Logan, Vermont). Hematoporphyrin dihydro-
chloride, protoporphyrin IX, and hemin (bovine type I) were
purchased from Sigma. Hematoporphyrin derivative (HPD) was
prepared by treating hematoporphyrin with a mixture of glacial
acetic acid and sulfuric acid in a ratio of 19:1 (vol/vol) and
processed as described previously [4].
Polycationic peptides and antibiotics. Poly-L-lysine (hydrobro-
mide) was obtained from Sigma. Polymyxin nonapeptide (PMNP)
was prepared from polymyxin B sulfate obtained from Sigma by a
process described previously [30] with minor modifications. Purity
of PMNP batches was examined on cellulose-coated aluminum foil
TLC plates. Imipenem was obtained from Merck, Sharp and
Dohme (West Point, Pennsylvania), and cefoxitin was purchased
from Sigma.
Fluorescence measurements. Fluorescence spectra were measured
on a digital spectrofluorometer (Perkin-Elmer Luminescence spec-
trometer model LS-50, interfaced to a data station 7500 computer).
The excitation wavelength was 405 nm, and the emission spectra
were recorded from 550 nm to 700 nm. Strains were grown in
Brucella broth supplemented with vitamin K1 for 48 h in an
anaerobic chamber at 37~ Cultures were washed twice with
0.85% NaC1 by centrifugation (12,000 g, 20 min) and resuspended
in 0.1 M phosphate saline buffer (pH 6.5). Relative fluorescence at
the emission band of 622 nm was calculated and corrected to a
standard number of bacteria (109 cells/ml).
Cell envelope preparation. Cells were grown for 72 h in Brucella
broth supplemented with 1 ixg/ml vitamin K1 and 1% pyruvic acid.
Growth was harvested by centrifugation (12,000g, 35 min), and the
pellet was resuspended in 0.05 M Tris-HCl buffer (pH = 7.5)
containing 0.01 M MgC12and 5 ixg/ml DNase. Cells were broken by
three passages through a French pressure cell (SLM Instruments
Inc., Urbana, Illinois) at 12,000 p.s.i. Cell lysate was centrifuged
(1200 g, 10 min) to remove unbroken cells. The supernatant was
again centrifuged (45,000g, 45 min), and the pellet (containing cell
envelope) was washed once with 0.05 M Tris-HC1 (pH = 7.5) and
used immediately.
Results
Photoinactivation studies were performed with Gram-
positive and Gram-negative strains of anaerobic bac-
t e r i a (350 strains). T h r e e h u n d r e d a n d s i x t e e n strains
w e r e s c r e e n e d for t h e i r s u s c e p t i b i l i t y to d e u t e r o p o r -
p h y r i n a n d light. T a b l e 1 lists t h e c u m u l a t i v e p e r c e n t -
a g e o f s t r a i n s i n h i b i t e d by p h o t o s e n s i t i z e d D P f o r
each group of microorganisms. Among the Gram-
n e g a t i v e a n a e r o b i c b a c t e r i a , all s t r a i n s of Porphyromo-
nas spp. a n d Prevotella spp. w e r e i n h i b i t e d by p h o t o -
s e n s i t i z e d D P ( M I C s o f < 2.5 jxg/ml). O f t h e s t r a i n s
o f B. fragilis, 9 0 . 4 % h a d a n M I C o f < 2.5 ~ g / m l , a n d
o n l y o n e s t r a i n ( o f 42) w a s n o t i n h i b i t e d by c o n c e n t r a -
t i o n s o f a b o v e 60 ~ g / m l D P a n d light. O n t h e o t h e r
h a n d , all B. gracilis, B. wadsworthia, F. mortiferum,
a n d F. varium strains w e r e n o t i n h i b i t e d by p h o t o s e n -
s i t i z a t i o n , e v e n w i t h D P at c o n c e n t r a t i o n s o f > 60
jxg/ml. B e t w e e n 9 2 . 3 % a n d 9 4 . 7 % o f t h e G r a m -
p o s i t i v e a n a e r o b i c c o c c i a n d r o d s ( e x c e p t for Clos-
Y. Nitzan et al.: Inactivation of Anaerobic Bacteria 127

Table 1. Cumulative inhibition of anaerobe species by photoactivated deuteroporphyrin

Strains inhibited at (p,g/ml) by DP and light: Strains not

inhibited by
No. of < 2.5 _<5 < 10 < 20 > 60 ixg/ml DP
strains
Microorganisms tested No. (%) No. (%) No. (%) No. (%) No. (%)

Gram-negative anaerobes:
Bacteroidesfragilis 42 38 (90.4) 38 (90.4) 40 (95.2) 41 (97.6) 1 (2.4)
Bacteroides thetaiotaomicron
anddistasonis 28 11 (39.3) 21 (75.1) 23 (82.2) 26 (92.9) 2 (7.1)
Bacteroidesgracilis 11 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 11 (100.0)
Other Bacteroides spp. 33 19 (57.6) 27 (81.8) 31 (94.0) 32 (97.0) 1 (3.0)
Bilophila wadsworthia 33 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 33 (100.0)
Fusobacterium mortiferum
and variurn 11 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 11 (100.0)
Other Fusobacterium spp. 22 15 (68.2) 15 (68.2) 15 (68.2) 16 (72.7) 6 (27.3)
Porphyromonas spp. 35 35 (100.0) 35 (100.0) 35 (100.0) 35 (100.0) 0 (0.0)
Prevotella spp. 32 32 (100.0) 32 (100.0) 32 (100.0) 32 (100.0) 0 (0.0)
Gram-positive anaerobes:
Clostridium spp. 24 18 (75.0) 21 (87.5) 21 (87.5) 22 (91.7) 2 (8.3)
Gram (+) cocci 26 24 (92.3) 24 (92.3) 26 (100.0) 26 (100.0) 0 (0.0)
Gram (+) rods 19 18 (94.7) 18 (94.7) 18 (94.7) 18 (94.7) 1 (5.3)

Table 2. Cumulative inhibition of anaerobes by hemin

Strains inhibited at v~g/mlby hemin Strains not

inhibited by
No. of < 10 < 20 < 40 > 60 p~g/mlheroin
strains
Microorganisms tested No. (%) No. (%) No. (%) No. (%)

Gram-negative anaerobes:
Bacteroidesfragilis 42 1 (2.4) 1 (2.4) 2 (4.8) 40 (95.2)
Other Bacteroides spp. 72 1 (1.4) 7 (9.7) 7 (9.7) 65 (90.3)
Bilophila wadsworthia 33 0 (0.0) 0 (0.0) 0 (0.0) 33 (100.0)
Fusobacterium spp. 33 5 (15.2) 7 (21.2) 7 (21.2) 26 (78.8)
Porphyromonas spp. 35 26 (74.3) 33 (94.3) 33 (94.3) 2 (5.7)
Prevotella spp. 32 5 (15.6) 11 (34.4) 16 (50.0) 16 (50.0)
Gram-positive anaerobes:
Clostridium spp. 24 2 (8.3) 5 (20.8) 10 (41.6) 14 (58.4)
Gram (+) cocci 26 19 (73.1) 20 (76.9) 24 (92.3) 2 (7.7)
Gram (+) rods 19 9 (47.4) 14 (73.7) 16 (84.2) 3 (15.8)

tridium) had an MIC of < 2.5 txg/ml DP. Only 75% of
Clostridium spp. were inhibited by DP (MIC < 2.5
ixg/ml), but the percentage of all Gram-positive
anaerobes that were not inhibited by more than 60
Ixg/ml DP (in the light) was very low. All the strains
that are photosensitized in the light by 2.5 ixg/ml DP
were able to grow even in the presence of 60 wg/ml
DP in the dark.
The MICs of heroin (Fe-bound porphyrin) for
316 strains were also tested. Cumulative percentages
of inhibited strains in each group of anaerobes are
listed in Table 2. Porphyromonas spp. and the Gram-
positive anaerobes (cocci and rods excluding Clos-
tridium spp.) were inhibited by hemin. For most of the
Porphyromonas strains (74.3%), the MICs were about
10 Ixg/ml. No strain was inhibited by 5 ixg/ml hemin
(hemin as a growth additive is normally added at 5
ixg/ml). Porphyromonas spp. are the only Gram-
negative anaerobic species shown to be significantly
inhibited by heroin. Anaerobic Gram-positive cocci
were also inhibited by hemin. Gram-positive rods
were less inhibited by heroin (47.4% were inhibited at
10 ixg/ml hemin, and 84.2% were inhibited at 40
ixg/ml). Thiolic agents, such as mercaptoethanol or
cysteine (> 10 -2 M), can reverse the inhibition by
hemin.
128 CURRENTMICROBIOLOGYVol. 29 (1994)

Table 3. Cumulative inhibition of Porphyromonas strains by different photoactivated porphyrins

Strains not in-

Strains inhibited at (~g/ml) by porphyrin and light hibited by > 60
o,g/ml photo-
sensitized
No. of <2.5 _<5 < 10 <20 <40 porphyrin
strains
Porphyrin tested No. (%) No. (%) No. (%) No. (%) No. (%) No. (%)
DP 35 35 (100) 35 (100) 35 (100) 35 (100) 35 (100) 0 (0.0)
HPD 31 4 (12.9) 4 (12.9) 9 (29.1) 14 (58.1) 20 (77.4) 7 (22.6)
HP 30 1 (3.3) 3 (9.9) 4 (13.2) 13 (43.2) 23 (76.6) 7 (23.4)
PP 29 0 (0.0) 2 (6.9) 3 (10.3) 3 (10.3) 7 (24.1) 22 (75.9)

622nm
6.25 - -
Porphyromonas 6.0- Bilophlla
100 - (P-32) (B-r)
5.5-
90- 622nm
S.0-

+ 80- i70--
' ; :
4.5-

|
-- i !~: i '.~
s+ i- /

f 8 ,/r+1, '-- /
40- !,.5-I., ../ , \,,.~j. ,
,.o- j+ .... /!
=o So- E
.~
' '-"'-. :is
.n- 2o- ,L~ 1.5- I ~'"~ .-. J/i

~i!~ 1'~ ..... "~"+

10- __

55~.0~u,, ~,v ~ ~ ..... ,, . . . . . . . . . . . . . . . . . 700 550.0 ,,v

Wavelength (nm) Wavelength (nm)
Fig. 1. Emission fluorescence spectra of DP (20 ixg/ml) with varying numbers of Porphyromonas cells (left panel) and Bilophila cells (right
panel) in 0.1 M phosphate-buffered saline at pH = 6.5; excitation wavelength was at 405 nm. (a) No organisms, DP only; (b) 3.9 • 10s;
(c) 7.8 • 108; (d) 1.2 • 109; (e) 1.6 x 109; (f) 2.4 • 109 cells per ml.

The photosensitization-killing of anaerobic bacte-
ria by various porphyrins was examined with strains of
Porphyromonas spp. Although Porphyromonas strains
are sensitive to photosensitization by DP, they are
less sensitive to HPD or HP (Table 3). The least
inactivation was seen with PP and light. These results
reflect the structural differences between these por-
phyrin molecules.
The fluorescence spectrum of DP was used to
interpret the different bindings of DP to Porphyromo-
nas strains (sensitive to DP and light) and Bilophila
strains (resistant to photosensitization by DP). The
fluorescence spectra of DP in the presence of varying
numbers of Porphyromonas or B. wadsworthia are
shown in Fig. 1. The fluorescence intensity at the 622
nm band increases concomitantly with the number of
Porphyromonas cells (Fig. 1, left panel). On the other
hand, the fluorescence intensity of B. wadsworthia at
the 622-nm band remains small even with high
numbers of bacterial cells (Fig. 1, right panel), indicat-
ing a markedly lower affinity for DP by Bilophila ceils
compared with Porphyromonas cells. With equivalent
numbers of the two bacteria (109 cells/ml), the
fluorescence intensities at 622 nm show a significant
difference (Fig. 2). The ability of Bilophila envelopes
to bind DP (Fig. 3) led us to try to expose these
binding sites in living cells. Pretreatment of Bilophila
with imipenem or cefoxitin allowed much better
binding of DP to Bilophila cells, as seen by the
fluorescence intensities (Fig. 3). Furthermore, only
Y. Nitzan et al.: Inactivation of Anaerobic Bacteria
120[ ~ [
100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
,,=,
50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
50
40 . . . . . . . . . . . .
=.:
20
0
5 6 9 112023253032
STRAIN NUMBER
Fig. 2. Relative fluorescence of DP (20 I~g/ml) emission at 622 nm
(excitation at 405 nm) with various Porphyromonas and Bilophila
strains. Intensities were corrected to the same number of cells (109
cells/ml).
by the synergistic effect of the antibiotic and photoac-
tivated DP could the Bilophila cells be killed by small
amounts of DP (10 Cg/ml). The polycationic pep-
tides, polymyxin nonapeptide and poly L-lysine, which
can expose Gram-negative aerobic bacterial enve-
lopes, failed to expose the envelopes in B. wadswor-
thia strains.
Discussion
We report here an extensive study of photosensitiza-
tion of anaerobes by porphyrins. Although the antimi-
crobial effects of iron-free porphyrins in their excited
state have already been established and reviewed
recently [15, 25], the knowledge of the effects of these
compounds on anaerobic bacteria has remained very
limited. Only four strains of Bacteroides fragilis, Clos-
tridium perfringens, Peptococcus magnus, and Pepto-
streptococcus anaerobius [32] were tested and found to
be inactivated by HPD and light. In the present study,
we employed 350 strains including most clinically
significant anaerobic species and Gram-negative rods
as well as Gram-positive rods and cocci. This investi-
gation revealed that the photodynamic effect of
porphyrins occurs in a large variety of anaerobic
bacteria. Most of the anaerobic species, both Gram
negative and Gram positive, are inactivated by light-
activated deuteroporphyrin (DP) at concentrations of
< 2.5 txg/ml. The latter concentration is at least four-
to fivefold lower than the concentrations needed for
aerobic bacteria [22, 26]. On the other hand, B.
wadsworthia, F. mortiferum, F. varium, and B. gracilis
were not inactivated even at concentrations of > 60
i~/ml DP. From the overall photoinactivation pattern
by DP of anaerobic bacteria, it seems that the
DP-inactivated species are also Gram-negative bacte-
ria. Furthermore, these DP-photoinactivated, Gram-
129
250
B-13 B-24
u., 200
0
Z
150
O
p
. . . . . . . . . . . . . ".* 100
" 511
1 3 7 121316242932
O
we ENV IM CEF NP PI. WC ENV IM CEF NP PL
Fig. 3. DP relative emission fluorescence at 622 nm (excitation at
405 nm) with two Bilophila wadsworthia strains grown in different
conditions: (A) Intact whole cells (WC), grown normally, served as
controls; (B) Bilophila cell envelopes (ENV); (C) Bilophila cells
grown with 100 ~g/ml imipenem (IM); (D) Bilophila cells grown
with 100 p~g/ml cefoxitin (CEF); (E) Bilophila cells grown with 250
ixg/ml polymyxin nonapeptide (NP); (F) Bilophila cells grown with
250 ~g/ml polylysine (PL).
negative anaerobes do not require membrane disorga-
nizing agents like polymyxin nonapeptide (PMNP). It
was shown previously that the photoinactivation of
aerobic bacteria is different [22, 26]. Only Gram-
positive aerobic bacteria are photoinactivated di-
rectly by DP and light [22]. Gram-negative aerobic
bacteria require the presence of PMNP for photosen-
sitization and inactivation. DP needs PMNP to en-
able it to penetrate through the bacterial barrier in
order to bind to the inner membrane [26] and to
produce the photodynamic effect and killing of the
cell. As indicated above, within the Gram-negative
anaerobic species there are two groups: (1) those
whose outer barrier permits the penetration of DP to
the inner membrane and which are photoinactivated
and (2) those whose barrier (probably the outer
membrane) is not permeable to DP and which are
therefore not photoinactivated. Surprisingly, the
Gram-negative anaerobic species that are not inhib-
ited by photosensitized DP are not affected even by
the presence of PMNP or polylysine and remain
uninhibited.
By the fluorescence parameters employed in this
study, we could differentiate the nature of the environ-
ment in which the DP molecule resides in the
microbial barriers. Measurements of the fluorescence
spectrum of DP (excitation at 405 nm) were used to
interpret the different binding ability of DP to Porphy-
romonas strains and to Bilophila strains. We found a
correlation between the extent of DP binding and the
observed photodynamic effect (killing of the cells) in
these two species. It is clear that Porphyromonas
strains bind enormous quantities of DP and conse-
130
quently are killed by the photodynamic process that
follows upon illumination. On the other hand, Bilo-
phila strains bind relatively small amounts of DP and
are not killed by the photosensitization reaction even
when high concentrations of DP are used. The band
at 622 nm is the typical emission of DP in a low-
polarity medium [15]. The low-polarity phase in the
bacterial cell that results in this band for DP is the
cytoplasmic membrane, as was previously shown for
aerobic Gram-positive [6] and for aerobic Gram-
negative bacteria (in the presence of PMNP) [26].
The possibility that the Bilophila cytoplasmic mem-
brane is not capable of binding DP was ruled out by
showing that the envelope preparation of Bilophila
strains can bind DP at least as well as intact Porphy-
romonas cells. Thus, disorganizing or disrupting the
bacterial barriers and exposing the inner membranes
will allow DP to bind. However, agents that disrupt
the outer membranes, such as PMNP or polylysine,
did not increase DP binding to Bilophila. It may be
that the Bilophila and other Gram-negative anaer-
obes have a more complicated outer membrane
structure or that they cannot be disrupted by the
polycationic agents. A recent review [28] indicated
that in anaerobic Gram-negative bacteria, PMNB or
polycationic agents do not increase permeability of
the outer membrane. Removal of the outer barriers
and exposure of the inner membrane was accom-
plished by the antibiotics imipenem and cefoxitin.
The presence of these antibiotics in the growth
medium causes the release of lipopolysaccharide
from the bacteria [7] (70-80% of the lipopolysaccha-
ride can be released in their presence). Only the
synergistic effect of imipenem or cefoxitin and DP
allowed the porphyrin to bind to the bacterial cell (to
the inner membrane) and to kill the cell upon
photosensitization. Again, the important role of the
binding of the photosensitizer molecule to the inner
membrane is indicated. Furthermore, there are por-
phyrin molecules other than DP (e.g., HPD, HP, and
PP) that do not photoinactivate these bacteria to the
same extent as DP. It was shown in this study that
Porphyromonas spp. are less inactivated by HPD or
HP and are still less inactivated by PP, indicating that
the inactivation by porphyrins may also depend on
the chemical structure of each molecule, which deter-
mines its ability to penetrate the bacterial barriers
and to reach the inner membrane.
The mechanism by which the porphyrin molecule
exerts its photodynamic effect and the major target
sites of inactivation within the bacterial cell are under
further investigation. From preliminary results it
seems that as a consequence of the anaerobic condi-
CURRENT MICROBIOLOGYVol. 29 (1994)
tions that exist during the time period of the photody-
namic process, the photochemical reaction taking
place is of the type I mechanism [14, 31]. Hydroxyl
free radical (OH-) and probably other free radicals
are produced and as a result kill the cells. In aerobic
bacteria singlet oxygen [102] and hydroxyl free radi-
cals inhibited the bacteria [24[.
Hemin, the iron-containing protoporphyrin, has
been shown to possess antibacterial activity that was
not dependent on illumination. Porphyromonas spp.,
Gram-positive cocci, and many Gram-positive rods
(excluding clostridia) were found to be inactivated by
hemin. This phenomenon was found previously with
Gram-positive aerobic bacteria [9, 21] but not with
Gram-negative aerobic cells. In the present study, the
'strains were shown to have an MIC to hemin of 10-20
~g/ml. This concentration is only two- to fourfold
higher than the amount of hemin recommended as a
growth additive for anaerobes (5 ~g/ml). Heroin
affects the cells not by photosensitization but rather
through an oxidation-reduction process in connection
with peroxide [14]. With anaerobic bacteria as with
aerobes [23], we found that thiol reagents can protect
from hemin inactivation. Thus, the antibacterial ef-
fect of hemin must be different from the antimicro-
bial effects of the metal-free porphyrins, where the
bacteria are not protected from killing by thiols. It
should be noted that excess exogenous heroin (as a
supplement) can lead to cell inactivation instead of
increasing growth.
The antibacterial action of porphyrins on anaer-
obes may ultimately have therapeutic potential. These
results suggest new possibilities for therapy of anaero-
bic bacterial infections. This modality is independent
of antibiotic resistance of the pathogen and can also
act synergisticallywith antibiotics to which the patho-
gen is resistant. Some of these porphyrins have
already been used for clinical treatments [17], and the
source of light can be achieved by optic fiber even in
body cavities. In addition, it seems from preliminary
results on the fluorescence of bound porphyrins to
bacteria that porphyrins may be good probes to define
subgroups in species found to be a single genus, such
as Bilophila wadsworthia [1].
ACKNOWLEDGMENTS
This work was supported in part by VA Merit ReviewMedical
Research Funds and in part by Fujisawa Pharmaceuticals Co.
(Deerfield,Illinois).
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