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Antimicrobial Agents and Chemotherapy, September 1998, p. 2290-2294, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vitro Inactivation of Chlamydia
trachomatis by Fatty Acids and Monoglycerides
Gudmundur
Bergsson,1
Jóhann
Arnfinnsson,2
Sigfús M.
Karlsson,3
Ólafur
Steingrímsson,3 and
Halldor
Thormar1,*
Institute of Biology, University of
Iceland,1
Department of Anatomy,
University of Iceland Medical School,2 and
Department of Microbiology, National University
Hospital,3 Reykjavik, Iceland
Received 9 February 1998/Returned for modification 16 March
1998/Accepted 29 June 1998
 |
ABSTRACT |
The antichlamydial effects of several fatty acids and
monoglycerides were studied by incubating Chlamydia
trachomatis bacteria with equal volumes of lipid solutions for 10 min and measuring the reduction in infectivity titer compared with that
in a control solution without lipid. Caprylic acid (8:0), monocaprylin
(8:0), monolaurin (12:0), myristic acid (14:0), palmitoleic acid
(16:1), monopalmitolein (16:1), oleic acid (18:1), and
monoolein (18:1) at concentrations of 20 mM (final concentration,
10 mM) had negligible effects on C. trachomatis. In
contrast, lauric acid (12:0), capric acid (10:0), and monocaprin
(10:0) caused a greater than 10,000-fold (>4-log10)
reduction in the infectivity titer. When the fatty acids and
monoglycerides were further compared at lower
concentrations and with shorter exposure times, lauric acid was more
active than capric acid and monocaprin was the most active,
causing a greater than 100,000-fold (>5-log10)
inactivation of C. trachomatis at a concentration
of 5 mM for 5 min. The high levels of activity of capric and lauric
acids and particularly that of monocaprin are notable and
suggest that these lipids have specific antichlamydial effects. The
mode of action of monocaprin was further studied by
removal of the lipid by centrifugation before inoculation of Chlamydia onto host cells and by electron microscopy. The
results indicate that the bacteria are killed by the lipid,
possibly by disrupting the membrane(s) of the elementary
bodies. A 50% effective concentration of 30 µg/ml was found by
incubation of Chlamydia with monocaprin for 2 h. The
rapid inactivation of large numbers of C. trachomatis organisms by monocaprin suggests that
it may be useful as a microbicidal agent for the prevention of the
sexual transmission of C. trachomatis.
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INTRODUCTION |
Chlamydia trachomatis is
the most common sexually transmitted bacterial pathogen. Annually,
there are an estimated 50 million new cases of C. trachomatis infection worldwide (4), with more than 4 million occurring in the United States and 3 million occurring in
Europe (2). Although they are treatable with antibiotics, many Chlamydia infections go undetected, particularly in
women, and can cause severe permanent damage to the female genital
tract, which may lead to infertility (18). A vaccine against
C. trachomatis has not been developed, and other means
of prevention except for the use of condoms are not available. In
recent years there has been considerable interest in the use of
microbicidal compounds for the prevention of sexually transmitted
diseases (STDs) (3). Vaginal spermicides, which have been
used as contraceptives for a number of years, have been shown to kill
sexually transmissible bacteria and viruses in vitro and in vivo
(13). The microbicidal activity of the nonionic surfactant
nonoxynol-9 has been studied most extensively. Two studies found that
nonoxynol-9 inactivates C. trachomatis in vitro
(1, 9), whereas one study found that it did not
(8). There is evidence from clinical trials that it may have
some effect against chlamydial infections in vivo (12).
However, because of the toxicity of nonoxynol-9 to mucosal membranes,
particularly when it is applied frequently, there is a need for other
less toxic microbicidal compounds which could be used to provide
protection against STDs (3).
The microbicidal effects of a variety of lipids have been
extensively studied in recent years. A number of free fatty acids and
their 1-monoglycerides have been found to kill enveloped viruses and
various bacteria, both gram-negative and gram-positive bacteria (5, 7, 14, 15, 17). These lipids are commonly found in
natural products, for example, in milk, and can therefore be assumed to
be nontoxic to mucosas, at least at low concentrations. It has
therefore been suggested that they might be useful as intravaginal microbicides for protection against STDs (6). In order to
prevent infections caused by sexually transmitted viruses, it is
important that the microbicidal lipid is fast acting and kills the
virus before it has time to infect cells of the genital mucosa. The same is true for bacteria such as Chlamydia, which,
like viruses, replicate intracellularly. In the present study
several fatty acids and their 1-monoglycerides which have previously
been found to inactivate enveloped viruses (15) were tested
for their microbicidal activities against C. trachomatis. A short inactivation time of 10 min or less
was selected as a criterion for a fast and
effective killing of the bacteria.
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MATERIALS AND METHODS |
Cell culture.
Monolayer cultures of McCoy cells, a
heteroploid mouse fibroblast cell line, were used for cultivation of
C. trachomatis and in antichlamydial assays. They were
grown in RPMI 1640 medium (GIBCO) containing 5% (vol/vol)
heat-inactivated fetal calf serum, 45 mM sodium bicarbonate, 2 mM
L-glutamine, and 0.05 mg of gentamicin per ml. This was
called the base medium (BM).
The cell cultures were maintained by weekly trypsinization and passage
in 260-ml tissue culture flasks (Nunclon). The cultures were passaged
in the following manner. The medium was removed and the monolayer was
rinsed twice with 10 ml of Hanks' balanced salt solution (GIBCO). The
monolayer was covered with 1 ml of trypsin-EDTA solution (GIBCO) and
was carefully shaken until the cells came into suspension. The trypsin
activity was then stopped by adding 10 ml of BM, and the cells were
evenly suspended by pipetting. They were counted, and 107
cells in 20 ml of BM were seeded into each flask. Cell cultures were
kept at 37°C in a humidified incubator with 5% CO2 in
air.
Chlamydial stock cultures.
C. trachomatis
serotype K, originally isolated from a human cervix, was obtained from
the American Type Culture Collection (strain VR 887). The concentration
of bacteria used in the experiments varied from 106.5 to
107.8 inclusion-forming units (IFU) per ml. Two strains of
C. trachomatis were recent clinical isolates. One was
isolated from a cervical swab, and the other was isolated from a
urethral swab from a male patient. They were subcultured once in McCoy
cells and were used at concentrations of 105.1 and
105.4 IFU per ml, respectively. All bacterial stocks were
kept frozen at 
70°C in a mixture of equal volumes of cycloheximide
medium (CM) and transport medium. The latter is composed of 0.2 M
sucrose buffer supplemented with 10% inactivated fetal calf serum.
Lipids.
Fatty acids and 1-monoglycerides were purchased from
Sigma Chemical Co., St. Louis, Mo. (purest grade). Stock solutions of 1 M were made in ethanol.
Assay of antichlamydial activity.
Twenty-four-well (16 mm in
diameter) tissue culture plates (Corning, Corning, N.Y.) were seeded
with 6 × 105 McCoy cells per well in 2 ml of BM.
Twenty-four hours later, when the cells had formed a confluent
monolayer, the medium was changed to 1 ml of maintenance medium (MM),
which is BM supplemented with 0.5% (wt/vol) glucose. Stock solutions
of fatty acids or monoglycerides were diluted to the desired
concentration by vortexing at the highest speed for 1 min at
37°C. The solutions showed a little turbidity which varied
between lipids but which was less for lipids with shorter fatty acid
chains. The solutions were immediately tested against C. trachomatis by thoroughly mixing together 100 µl of a lipid
solution and 100 µl of the bacterial stock in a plastic tube. The
mixtures were incubated for the desired time at 37°C. Samples
were removed and diluted in MM in 10-fold dilutions, and 0.2 ml of each
dilution was inoculated into culture plates with McCoy cells (four
wells for each dilution). Bacteria mixed with MM alone and with 2%
ethanol in MM were used as controls. The plates were centrifuged at
1,100 × g for 75 min at 35°C and were then incubated
for 2 h at 37°C in an atmosphere of 5% CO2. Finally, the medium was replaced with 1 ml of MM containing 2 µg of
cycloheximide per ml (CM) to inhibit protein synthesis of the host
cells, and the incubation was continued for an additional 72 h.
The CM was then removed from each well and the monolayers were fixed
with 96% ethanol and stained with iodine-glycerol stain for 10 min.
Brown inclusions filled with Chlamydia could be
distinguished in the cytoplasms of infected cells when the cells were
viewed under a microscope.
In a few experiments inclusions were stained with fluorescein-labeled
monoclonal antibodies specific for C. trachomatis. A C. trachomatis culture confirmation test (Syva
Microtrak; Syva Company, San Jose, Calif.) was performed by the
procedure described by the manufacturer.
Inclusions were counted in all four wells of the dilution which
contained 10 to 50 inclusions per well. The average number
of
inclusions in that dilution was evaluated, and the number of
IFU per
milliliter of undiluted sample was calculated. The number
(log
10) of IFU in the lipid-treated sample was subtracted
from
the number (log
10) of IFU in the control sample, and
the difference
was used as a measure of antichlamydial activity, i.e.,
the ratio
of bacteria inactivated by incubation with a lipid solution
for
a given length of time at 37°C.
Because of toxicity to cell cultures, lipid-bacterium mixtures could
not be tested undiluted or at a dilution of 10
1,
depending on the lipid or the lipid concentration. In such
lipid-bacterium
mixtures in which no inclusions were detectable in the
10
1 dilution, it was assumed that the undiluted mixture
contained
45 IFU per ml, i.e.,
1.7 log
10 IFU.
Similarly, when no inclusions
were detectable in the 10
2
dilution, the undiluted mixture might contain
450 IFU per ml,
or
2.7 log
10 IFU. The antichlamydial activity was therefore
calculated
by subtracting 1.7 or 2.7, respectively, from the
log
10 number
of IFU for the control sample, and the numbers
of IFU expressed
the activity as being equal to or greater than this
difference.
Washing of monocaprin-treated Chlamydia.
One
milliliter of bacterial stock was diluted fivefold with antibiotic-free
BM (a-BM) and was pelleted by centrifugation at 2,000 × g in a Sorvall RT 6000D centrifuge at 35°C for 30 min, and
the bacterial pellet was resuspended in 1 ml of a-BM. The bacteria were
mixed with an equal volume (200 µl) of 10 mM monocaprin in a-BM, and
the mixture was incubated at 37°C for both 5 and 10 min. Bacteria
incubated in the same way with a-BM without lipid served as a control.
After incubation each mixture was diluted 10-fold and the dilution was
divided into two samples. One sample was kept at 35°C for the
duration of the experiment, whereas the bacteria in the other sample
were washed twice by centrifugation at 2,000 × g at
35°C for 30 min and the pellet was resuspended in 2 ml of a-BM. The
infectivity titers of all the samples were then determined by
inoculation of 10-fold dilutions onto McCoy cells in 24-well culture
plates as described above.
Electron microscopy.
A suspension of C. trachomatis was mixed with an equal volume of 10 mM monocaprin in
MM, and the mixture was incubated at 37°C. Samples were removed
after 1, 5, and 10 min and were immediately diluted 1:10 in MM.
Chlamydiae incubated for 10 min with an equal volume of MM without
monocaprin were diluted 1:10 and were used as a control. Three hundred
microliters of each sample was pipetted into two wells of a 96-well
microtiter plate, and the plates were centrifuged at 1,100 × g for 60 min at 35°C so that the samples adhered to
Formvar-coated 300-mesh copper grids which had been placed on the
bottoms of the wells. After centrifugation the grids were removed, and
the bacteria were negatively stained with 1% phosphotungstic acid (pH
7.0) and examined in Philips 300 transmission electron microscope at 80 kV.
Assay of the MIC of monocaprin.
Ninety-six-well microtiter
plates (Nunclon) were seeded with 105 McCoy cells per well
in 100 µl of BM. Twenty-four hours later the cells had formed a
confluent monolayer and were ready for inoculation with the
Chlamydia. Monocaprin was diluted at 37°C by vortexing at
a high speed so that after the addition of the chlamydial culture at a
final concentration of 104 to 105 IFU per ml
monocaprin concentrations of 2.5 to 2,500 µg/ml were achieved. The
dilutions were tested against the bacteria by thoroughly mixing
together 200 µl of a monocaprin dilution and 200 µl of the
bacterial suspension. After 2 h of incubation at 37°C, the monocaprin concentrations were reduced by a 100-fold dilution in MM.
Then, 200 µl of each diluted mixture was inoculated into six wells of
a microtiter plate with McCoy cells. Bacteria mixed with MM served as a
control. The plates were centrifuged at 1,100 × g for
75 min at 35°C and were incubated for 2 h at 37°C in an atmosphere of 5% CO2. The medium was then replaced with
200 µl of CM. The cultures were incubated for 72 h before
staining with iodine, and the numbers of IFU were counted as described
above. The same concentrations (2.5 to 2,500 µg/ml) of monocaprin
were tested for cytotoxicity with monolayers of McCoy cells. Cell
viability was determined after 2 h at 37°C by trypan blue
exclusion.
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RESULTS |
Activities of fatty acids.
A comparison of the antichlamydial
activities of six fatty acids is presented in Fig.
1. The open bars represent the reduction in infectivity titers (log10 IFU) of C. trachomatis after incubation with an equal volume of 20 mM fatty
acid for 10 min at 37°C. Caprylic acid (8:0), myristic acid
(14:0), and the unsaturated fatty acids palmitoleic (16:1) and oleic
acids (18:1) did not cause a significant inactivation of the bacteria,
with the reduction in titer varying from zero to 0.3 log10.
In contrast, capric acid (10:0) and lauric acid (12:0) reduced the
titer by greater than 10,000-fold (
4 log10).
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FIG. 1.
Inactivation of C. trachomatis by
incubation with equal volumes of 20 mM fatty acids and monoglycerides
for 10 min at 37°C. The bars represent the levels of reduction
of the log10 numbers of IFU. The levels of inactivation by
capric acid and monocaprin (10:0) and by lauric acid (12:0) are
greater than those indicated by the bars (>4 log10). Two
percent ethanol had no effect on the infectivity titer.
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|
To compare the activities of capric and lauric acids, they were
tested at lower concentrations and with shorter incubation
times. The
data in Table
1 indicate that capric
acid lost most
of its activity when it was diluted to a concentration
of 10 mM,
whereas lauric acid was still very active at that
concentration,
causing a 500,000-fold reduction in titer (5.7 log
10). Lauric
acid was therefore more active than
capric acid against
C. trachomatis.
This was
confirmed by testing lauric acid at 5 mM, which caused
a 2,000-fold
(3.3 log
10) reduction in infectivity titer. Both
acids were
active at 20 mM for 5 min, but incubation for 1 min
had no effect.
Activities of monoglycerides.
The antichlamydial activities of
five 1-monoglycerides were tested, with monomyristin (14:0) being
omitted. The results are presented in Fig. 1. Monocaprylin (8:0),
monopalmitolein (16:1), and monoolein (18:1) had no significant
effect, and monolaurin (12:0) caused only a threefold reduction in
titer (0.5 log10). Monocaprin, on the other hand,
caused 40,000-fold or greater reductions in the infectivity titer of
C. trachomatis (4.6 log10). The high level of antichlamydial activity of monocaprin was confirmed by lowering the concentration to 10 and 5 mM and the incubation time to 5 min. As indicated in Table 2,
monocaprin was still active at 5 mM with an incubation
time of 5 min, causing a greater than 100,000-fold reduction in the
infectivity titer (5.1 log10). The high level of
antichlamydial activity of monocaprin was further confirmed by
staining with fluorescein-labeled monoclonal antibodies specific for
C. trachomatis. Monocaprin-treated
Chlamydia samples showed no inclusions at the
lowest dilution tested (101), whereas
control samples without monocaprin showed fluorescent Chlamydia inclusions at a dilution of 106 and
had a final infectivity titer of 107.3 IFU per ml. The
reduction in titer was therefore 400,000-fold or greater (5.6
log10). In order to show that the antichlamydial activity
of monocaprin was not limited to the one bacterial strain obtained from the American Type Culture Collection, two C. trachomatis strains recently isolated from patients were also
tested. Both strains were inactivated by monocaprin at a
concentration of 10 mM and incubation for 5 min, with the reductions in
titer being 3.4 and 3.7 log10, respectively.
In another experiment the lipid was removed by centrifugation and the
bacteria were washed in culture medium before inoculation
of 10-fold
dilutions onto cell monolayers. The results are presented
in Table
3. By comparing the titers
(log
10 numbers of IFU) of
the untreated control samples
that were not washed with those
in samples that were washed, it can be
seen that most of the bacteria
were recovered after washing, that is,
recoveries of 100 and 50%
in the samples obtained at 5 and 10 min, respectively. As expected
from previous experiments (Table
2), no
inclusion-forming bacteria
were detected in the 1:10 dilution of
unwashed samples after treatment
with monocaprin for 5 and 10 min. Thus, the loss of infectivity
was about 100,000-fold or greater,
i.e.,
4.9 log
10 IFU after
5 min and
5.0
log
10 IFU after 10 min. The same result was obtained
after
washing of the bacteria treated with monocaprin for 10 min.
In
this sample no inclusion-forming bacteria were observed in
the 1:10
dilution. The titer was therefore
1.7 log
10 IFU per
ml,
whereas the titer in the washed untreated control was 6.4
log
10 IFU per ml. On the other hand, in the sample
treated with
monocaprin for 5 min, inclusion-forming
bacteria became detectable
in the 1:10 dilution after washing. The
sample incubated for 5
min still showed an 8,000-fold (3.9 log
10) reduction in infectivity
titer compared to that for
the untreated control.
Electron microscopy of monocaprin-treated
Chlamydia.
Figure 2 shows
electron micrographs of elementary bodies (EBs) of C. trachomatis treated with 10 mM monocaprin for 1, 5, and 10 min. Untreated bacteria are shown for comparison (Fig. 2A). Bacteria
treated for 1 and 5 min (Fig. 2B and C, respectively) are not visibly
different from the untreated bacteria. On the other hand, after
treatment for 10 min (Fig. 2D) the EBs appear deformed and shrunken,
and there is some indication that some of them are disintegrating (Fig.
2D, inset). At a lower magnification it could be seen that in both
treated and untreated samples the bacteria are well dispersed on the
grid and there is no aggregation of EBs (data not shown).
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FIG. 2.
Electron micrographs of negatively stained EBs of
C. trachomatis. The EBs were untreated (A) and treated
with 10 mM monocaprin for 1 min (B), 5 min (C), and 10 min (D).
After treatment for 10 min, the EBs appear deformed and shrunken or
partially disrupted (D, inset). Bars, 1 µm.
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MICs.
The effects of a series of monocaprin
concentrations on Chlamydia upon contact for 2 h at
37°C are presented in Table 4. A
concentration of 50 µg per ml (0.2 mM) caused a 96% inactivation of
the bacteria, whereas after treatment with 25 µg per ml, 38% of the
bacteria were unable to form inclusions. There was less inactivation at
lower concentrations. The 50% effective concentration was about 30 µg per ml. Monocaprin at a concentration of 100 µg per ml
caused complete lysis of the cell layers in 2 h, whereas at 50 µg per ml and lower concentrations, no lysis was observed by the
trypan blue exclusion test. The bacteria were therefore about 2.5 times
more sensitive to the lipid than the host cells. It should be noted
that mixtures of Chlamydia and monocaprin were diluted 100-fold before inoculation onto McCoy cells. In the
tests, the cell monolayers were therefore exposed to
monocaprin at concentrations of 25 µg per ml or less.
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DISCUSSION |
Previous studies have shown that medium-chain saturated fatty
acids and long-chain unsaturated fatty acids and their
1-monoglycerides are potent inhibitors of enveloped viruses and
bacteria, both gram-negative and gram-positive bacteria (5,
14). In this study we have shown that C. trachomatis, a sexually transmitted, gram-negative bacterium, is
effectively inactivated by exposure for 10 min to 10 mM (final
concentration) lauric acid, a 12-carbon saturated fatty acid (12:0),
and to capric acid (10:0) and its 1-monoglyceride. The
monoglyceride of lauric acid had much less of an effect, and a number
of other fatty acids and their monoglycerides, i.e., caprylic
acid (8:0), myristic acid (14:0), and the unsaturated fatty acids
palmitoleic acid (16:1) and oleic acid (18:1), had no effect or only a
negligible effect. The narrow range of activities of the fatty acids
and monoglycerides against Chlamydia is notable and suggests
that these lipids have specific antichlamydial effects. A somewhat
wider range and higher levels of activity of fatty acids and
monoglycerides have been found against herpes simplex virus type 1 (HSV-1), against which, in addition to monocaprin and lauric
acid, palmitoleic acid and, to some extent, oleic acid cause a rapid
inactivation of the virus (10). Capric acid, on the other
hand, had no activity against HSV-1 under the same conditions.
The question of how monocaprin inactivates the infectivity of
Chlamydia was addressed by studying whether or not removal
of the lipid before inoculation into cell cultures restored the
infectivity. The results (Table 3) indicate that the loss of
infectivity was not caused by an effect of the lipid on host cells and
that the viability of the bacteria was irreversibly lost by treatment
with monocaprin. After treatment for 5 min a small viable
fraction became detectable by washing, showing that the bacteria were
not fully inactivated at this time. This is in agreement with the electron microscopy study (Fig. 2), which showed no visible changes in
the EBs after treatment with 10 mM monocaprin for 5 min, whereas after 10 min the EBs appeared deformed and partly
disintegrated. We therefore hypothesize that the lipid kills the
bacteria by affecting the outer membrane, leading to disruption of the
membrane(s) in 5 to 10 min. This is supported by a previous electron
microscopy study of the effect of linoleic acid on vesicular stomatitis
virus and on Vero cells, in which the viral envelope and the cellular membrane were disrupted by the fatty acid (15).
In a recent study (11) ethers of 6- and 8-carbon fatty acids
were tested against C. trachomatis.
2-O-Octylglycerol, which was the most active of the four
ethers tested, caused a complete inactivation of the bacteria in 2 h at a concentration of 7.5 mM but was apparently not fully active at
lower concentrations or with shorter exposure times. In our study we
chose to use short exposure times of 10, 5, or 1 min in order to detect
the rapid inactivation of Chlamydia. Monocaprin at a
final concentration of 2.5 mM reduced the infectivity titer by >5
log10 IFU in 5 min at 37°C and was the most active of all
the lipids tested in this study (Table 2). An exposure time of 1 min
had only a minor effect. This is in contrast to the case for HSV-1,
which is inactivated 100,000-fold or more (5 log10) by
exposure to monocaprin for 1 min (10). The rapid in
vitro killing of large numbers of sexually transmitted bacteria and
viruses by microbicides is an essential prerequisite for their possible
use in the prevention of STDs. Even if the number of infectious
bacteria or viruses transmitted to genital mucosas is several orders of
magnitude lower than the number used in in vitro tests, the conditions
for the killing of the microbe in vivo are likely to be less favorable
due to less effective mixing with the microbicide and an uneven
distribution of the microbe on the mucosa. A large margin of
microbicidal activity is therefore necessary. Due to the high in vitro
efficacy of monocaprin against C. trachomatis
and sexually transmitted enveloped viruses, this lipid may be useful as
a microbicidal agent for the prevention of the transmission of STDs.
Pharmaceutical formulations which contain monocaprin as the
active ingredient have been developed and are potent inactivators of
C. trachomatis, HSV-2, and human immunodeficiency virus
in vitro (16). In vivo testing of these formulations is
needed to establish whether or not they may be used for the prevention
of STDs.
Several studies have shown that spermicides such as nonoxynol-9
inactivate C. trachomatis and other sexually
transmitted bacteria and viruses (13), and it has been
suggested that they may be used for protection against STDs. Benes and
McCormack (1) studied the effects of various concentrations
of nonoxynol-9 on large numbers of C. trachomatis upon
contact for 120 min and found a 50% inhibition of inclusion formation
at a concentration of between 500 and 1,000 µg per ml. In a
comparable study of the activity of monocaprin against
Chlamydia, a greater inhibitory effect was found (Table 4),
with a 50% effective concentration of about 30 µg per ml. Although
toxic in cell cultures, monocaprin at a concentration of 5 mg
per ml (20 mM) has been shown not to cause irritation of the vaginal
mucosa of mice and rabbits (16). The low level of
toxicity in vivo and the high level of antichlamydial activity in vitro
suggest that monocaprin may be more useful than nonoxynol-9 as
protection against chlamydial infections.
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ACKNOWLEDGMENT |
This work was supported by a grant from the Research Fund of the
University of Iceland.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biology, University of Iceland, Grensasvegur 12, 108 Reykjavik,
Iceland. Phone: 354-525 4602. Fax: 354-525 4069. E-mail:
halldort{at}rhi.hi.is.
| |
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Abstract
Introduction
