# Spermicidal Activity of the Safe Natural  Antimicrobial Peptide Subtilosin

Authors: Katia E. Sutyak; Robert A. Anderson; Sara E. Dover; Kenneth A. Feathergill; Alla A. Aroutcheva; Sebastian Faro; Michael L. Chikindas
Journal: Infectious Diseases in Obstetrics and Gynecology
 (2008)

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## Abstract

Bacterial vaginosis (BV), a condition affecting millions of women each year, is primarily caused by the gram-variable organismGardnerella vaginalis. A number of organisms associated with BV cases have been reported to develop multidrug resistance, leading to the need for alternative therapies. Previously, we reported the antimicrobial peptide subtilosin has proven antimicrobial activity against G. vaginalis, but not against the tested healthy vaginal microbiota of lactobacilli. After conducting tissue sensitivity assays using an ectocervical tissue model, we determined that human cells remained viable after prolonged exposures to partially-purified subtilosin, indicating the compound is safe for human use.  Subtilosin was shown to eliminate the motility and forward progression of human spermatozoa in a dose-dependent manner, and can therefore be considered a general spermicidal agent. These results suggest subtilosin would be a valuable component in topical personal care products aimed at contraception and BV prophylaxis and treatment.


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## Body

## 1. Introduction

Subtilosin
is a ribosomally-synthesized  cyclopeptide produced byBacillus subtilis ATCC 6633 and  a recently identified natural isolate  of Bacillus amyloliquefaciens [1, 2]. This protein has a unique structure
among bacteriocins [3], and possesses antimicrobial activity
against a variety of pathogenic organisms, including Gardnerella vaginalis, Listeria
monocytogenes, and Streptococcus
agalactiae (Group B Streptococcus) [2]. The ability to
inhibit the growth of G. vaginalis is
of particular importance as it is one of the primary causative agents for
bacterial vaginosis (BV), a common condition found in up to 30% of women in
North America [4]. BV is characterized by the replacement
of normal vaginal lactobacilli with anaerobic bacteria (e.g., Prevotella, Bacteroides, and Mobiluncus)
and mycoplasmas, as well as a several log increase in overall bacterial growth
[5–7]. BV is asymptomatic in approximately one half of all cases, but is
associated with a wide variety of symptoms and problems ranging from
complications with pregnancies (i.e., preterm births, low fetal birth weight) to the development of pelvic
inflammatory disorder [7–11].
Recent studies have estimated that nearly one in three women in the United States
(29.2%) suffer from BV, with varying prevalence according to ethnicity and
education levels [4].
One of the most troubling aspects of BV infection is the association
with an increased risk of several sexually transmitted diseases (STDs),
including chlamydia, herpes, gonorrhea, trichomoniasis, and HIV/AIDS
[12–16].  The treatments recommended by the Centers for
Disease Control (CDC) are clindamycin and metronidazole administered either
orally or intravaginally [17].  Following these guidelines successfully
treats 60% of BV cases, but 20% of these cases return with highly developed antibiotic
resistances [18–20]. In such cases, it
would be extremely desirable to have an alternative form of treatment that
could fully eradicate the infection.Bacteriocins are typically divided
into two major, yet diverse, classes: class I, or lantibiotics, contain unusual
amino acids and are subject to extensive posttranslational modifications; and
class II, the heat stable nonlantibiotics [21]. Subtilosin has
a unique posttranslational structure that is unmatched among bacteriocins,
which has led to speculation that it may belong in a distinct and separate
class [2, 3]. Bacteriocins have been
widely considered for use in medicinal and pharmaceutical applications,
particularly for their bactericidal activity against multidrug resistant
organisms [22]. They are especially appealing alternatives
since their cost of production is so comparatively low to conventional
pharmaceutical treatments. For example, commercial grade nisin costs
approximately $100/lb through chemical distributors. Much attention has
recently been focused on the spermicidal activity of bacteriocins due to their
targeted antibacterial activity and lack of effect on human tissues. Nisin A, a
well-studied class I bacteriocin produced by Lactococcus lactis subsp. lactis [23], has human spermicidal activity [24, 25], and subtilosin has spermicidal activity against boar, bovine,
horse, and rat spermatozoa [26]. The results of these previous
studies suggest that subtilosin may also have spermicidal activity against
human spermatozoa, prompting our investigation.Since subtilosin has proven
antimicrobial activity against the pathogen largely responsible for BV, its
toxicity to human tissues was assayed to determine its potential as a safe
alternative remedy. For the human in
vitro study, the EpiVaginal ectocervical tissue model (MatTek
Corporation, Ashland, Mass, USA) was employed to examine the
relationship between prolonged exposure to subtilosin and cell viability. Spermicidal
evaluations were conducted to investigate the ability of the peptide to
restrict or eliminate sperm mobility, leading to its classification as a
spermicidal agent.


## 2. Materials and Methods

### 2.1. Production of Subtilosin

Subtilosin
was prepared as previously described [2]. To prepare a cell-free
supernatant (CFS), cells were removed by centrifugation (Hermle Z400K; LabNet,
Woodbridge, NJ, USA) for 25 minutes at 4500 g and 4°C. The supernatant was filter sterilized using 0.45μm filters (Fisher, Pittsburgh,  Pa, USA).
The protein of interest was precipitated from the supernatant by adding 30%
ammonium sulfate (w/v) while stirring overnight at 4°C and was resuspended in
20 mL of double distilled H2O. The column chromatography method
described by Sutyak et al. [2] was used to purify subtilosin from the CFS,
producing a near-pure isolate in the 90% methanol eluate. The antimicrobial
activity of all samples was confirmed by the well-diffusion assay according to
the protocol of Cintas et al. [27] with additional modifications [2]. The active fraction was concentrated to dryness using a Savant
SC110 Speed Vac and UVS400 Universal Vacuum System (Savant Instruments, Farmingdale, NY, USA), then
resuspended in 1.5 mL ddH2O.


### 2.2. Determination of Protein Concentration

#### 2.2.1. Column-Purified Sample Concentration

The concentration of subtilosin in the
column-purified fraction was determined using the Micro BCA Protein Assay Kit
according to the manufacturer’s protocol (Pierce, Rockford, Ill, USA).  In brief, the assay measures the reduction ofCu2+ to Cu1+ by colorimetric detection of Cu1+ by bicinchoninic acid. Bovine serum albumin (BSA) was used to develop a
standard curve with concentrations ranging from 0.5 to 20μg/mL; the concentration of subtilosin was calculated using the R value
from the trendline of the standard curve graph.


#### 2.2.2. Assay of Subtilosin Concentration in CFS

The concentration of subtilosin in the CFS was not
measurable with the Micro BCA Protein Assay due to the high level of background
proteins in the solvent (MRS medium). As an alternative, the protein concentration
was calculated by comparing the antimicrobial activity of known concentrations
of column-purified protein to equal volumes of CFS. Five two-fold dilutions
were made from the stock samples of both the CFS and the column-purified
fraction. Well diffusion assays were performed using50μL of each dilution against Micrococcus
luteus ATCC 10420, which is commonly used as a reference microorganism for
the determination of a bacteriocin’s biological activity [28].


### 2.3. Determination of the Presence of Weak  Organic Acids

As
reported previously, the concentration of lactic acid in the CFS was measured
to assess its potential effects on antimicrobial activity and cell viability
[2]. The quantity of each form of the acid in the sample was
measured using a commercially available D-lactic
acid/L-lactic acid kit (Roche
Boehringer, Mannheim, Germany), according to the
manufacturer’s instructions.


### 2.4. EpiVaginal Ectocervical Tissue Model

The
EpiVaginal (VEC-100) ectocervical tissue model (MatTek Corporation, Ashland, Mass, USA) was used and maintained as
fully described by Dover et al. [29]. The tissues were exposed to 83μL of subtilosin CFS (∼136μg/mL) for 4, 24, and 48 hours. For exposure times over 24 hours, the
tissues were aerated by placing them on two metal washers (MatTek Corporation, Ashland, Mass, USA) and fed with 5 mL of the assay
medium. Double-distilled water (ddH2O) was used as a negative
control, and was applied to cells after 6, 24, and 48 hours. A spermicidal
product containing 4% Nonoxynol-9 (Ortho Options CONCEPTROL Vaginal
Contraception Gel, Advanced Care Products, Skillman, NJ, USA) was used as a positive control
based on its documented cytotoxic properties [30, 31]. A cream (Monistat-3, Ortho
McNeil  Pharmaceutical, Inc., Raritan, NJ, USA) containing 4% of the nontoxic,
BV-active compound miconazole nitrate [30, 31], was used as a negative control.Following the designated exposure
times, the MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide) assay was used to determine overall cell viability. The data were used
to approximate an effective time (ET) that would reduce cell viability to 50%
(ET-50).


### 2.5. MTT Viability Assay

The
MTT assay was carried out according to the protocol outlined by Dover et al. [29].
Briefly, the viability of ectocervical cells after exposure to subtilosin was
measured as a direct proportion of the breakdown of the yellow compound
tetrazolium to the purple compound formazan, since only living cells can cause
this reaction to occur [32]. Tissues were exposed to subtilosin and
the two controls for several designated time points; at the conclusion of each,
the liquid in the plate wells was combined with the liquid from the tissue
inserts. This mixture was then assayed spectrophotometrically using a 96 well-plate
reader (MRX revelation, Dynex Technologies, Va, USA) to determine the level of
tetrazolium degradation.The viability (%) of the treated
tissue inserts was calculated according to an equation provided by the
manufacturer:%viability=OD570 (treated tissue)/OD570 (negative control tissue). The exposure time that reduced tissue viability by
50% (ET-50) was calculated according to [V=a+b*log(t)]
described by Ayehunie et al. [30], where V=%viability, t=time in minutes, and “a” and “b” are constants representing the viability data from the time points
preceding and following 50% viability. On the whole, there is a direct
relationship between the length of the ET-50 and the toxicity of the tested
application (i.e., a shorter ET-50 corresponds to a more harmful compound).


### 2.6. Semen Sample Collection and Analysis

The
CFS gathered from aB. amyloliquefaciens culture was used to test the effect of subtilosin exposure on the motility of
human spermatozoa. Initially, the CFS was diluted with normal saline (0.9%) so
that 200μL of the final material was equivalent to 50μL, 100μL, or 200μL of undiluted CFS.Two semen samples were collected on
the day of experimentation. Each sample was collected by self-masturbation in a
polypropylene specimen container (Fisher) prior to transport to the laboratory.
Within 1 hour of collection, the samples were pooled. Total sperm count was
calculated using bright field light microscopy (Olympus BX50; 400x) after
dilution (1:50) of the semen in normal saline. The initial percentage of motile
sperm was calculated prior to testing with a neubauer hemacytometer [33]. The determination of motile sperm % was made using randomly
selected field views (400x) from a count of between 104–201 cells. Any
visibly moving spermatozoa (directional or stationary) were counted as  motile cells.The percentage of forward
progressing spermatozoa was subjectively determined based on the assumption
that 70% of the sperm in a normal sample would behave in such a manner. The
samples used in this experimentation fell into such a “normal” category.


### 2.7. Treatment of Spermatozoa with Subtilosin

A modified
Sander-Cramer test was used to determine the effect of column-purified subtilosin
on human spermatozoa motility [34]. This measured the effect
of subtilosin after 30-second exposure times of 5 volumes (200μL) of the solution at each dilution (25% and 50% in
normal saline, and 100%) with one volume (40μL) of whole semen. The motilities of cells from random high-magnification
fields (400x) of the sample were determined in duplicate as described above.


### 2.8. Data Analysis

The % motility data were arcsine transformed
[35] prior to further examination. StatMost32 (version 4.1)
statistical software (DataMost Corporation, Sandy, Utah, USA) was used to calculate all
statistical parameters. The % values of motility were presented as averages and
90% confidence limits. Any differences between treatment groups were assessed
by the Newman-Keuls multiple range test. Differences were deemed significant at
the 0.05 level of confidence.


## 2.1. Production of Subtilosin

Subtilosin
was prepared as previously described [2]. To prepare a cell-free
supernatant (CFS), cells were removed by centrifugation (Hermle Z400K; LabNet,
Woodbridge, NJ, USA) for 25 minutes at 4500 g and 4°C. The supernatant was filter sterilized using 0.45μm filters (Fisher, Pittsburgh,  Pa, USA).
The protein of interest was precipitated from the supernatant by adding 30%
ammonium sulfate (w/v) while stirring overnight at 4°C and was resuspended in
20 mL of double distilled H2O. The column chromatography method
described by Sutyak et al. [2] was used to purify subtilosin from the CFS,
producing a near-pure isolate in the 90% methanol eluate. The antimicrobial
activity of all samples was confirmed by the well-diffusion assay according to
the protocol of Cintas et al. [27] with additional modifications [2]. The active fraction was concentrated to dryness using a Savant
SC110 Speed Vac and UVS400 Universal Vacuum System (Savant Instruments, Farmingdale, NY, USA), then
resuspended in 1.5 mL ddH2O.


## 2.2. Determination of Protein Concentration

### 2.2.1. Column-Purified Sample Concentration

The concentration of subtilosin in the
column-purified fraction was determined using the Micro BCA Protein Assay Kit
according to the manufacturer’s protocol (Pierce, Rockford, Ill, USA).  In brief, the assay measures the reduction ofCu2+ to Cu1+ by colorimetric detection of Cu1+ by bicinchoninic acid. Bovine serum albumin (BSA) was used to develop a
standard curve with concentrations ranging from 0.5 to 20μg/mL; the concentration of subtilosin was calculated using the R value
from the trendline of the standard curve graph.


### 2.2.2. Assay of Subtilosin Concentration in CFS

The concentration of subtilosin in the CFS was not
measurable with the Micro BCA Protein Assay due to the high level of background
proteins in the solvent (MRS medium). As an alternative, the protein concentration
was calculated by comparing the antimicrobial activity of known concentrations
of column-purified protein to equal volumes of CFS. Five two-fold dilutions
were made from the stock samples of both the CFS and the column-purified
fraction. Well diffusion assays were performed using50μL of each dilution against Micrococcus
luteus ATCC 10420, which is commonly used as a reference microorganism for
the determination of a bacteriocin’s biological activity [28].


## 2.2.1. Column-Purified Sample Concentration

The concentration of subtilosin in the
column-purified fraction was determined using the Micro BCA Protein Assay Kit
according to the manufacturer’s protocol (Pierce, Rockford, Ill, USA).  In brief, the assay measures the reduction ofCu2+ to Cu1+ by colorimetric detection of Cu1+ by bicinchoninic acid. Bovine serum albumin (BSA) was used to develop a
standard curve with concentrations ranging from 0.5 to 20μg/mL; the concentration of subtilosin was calculated using the R value
from the trendline of the standard curve graph.


## 2.2.2. Assay of Subtilosin Concentration in CFS

The concentration of subtilosin in the CFS was not
measurable with the Micro BCA Protein Assay due to the high level of background
proteins in the solvent (MRS medium). As an alternative, the protein concentration
was calculated by comparing the antimicrobial activity of known concentrations
of column-purified protein to equal volumes of CFS. Five two-fold dilutions
were made from the stock samples of both the CFS and the column-purified
fraction. Well diffusion assays were performed using50μL of each dilution against Micrococcus
luteus ATCC 10420, which is commonly used as a reference microorganism for
the determination of a bacteriocin’s biological activity [28].


## 2.3. Determination of the Presence of Weak  Organic Acids

As
reported previously, the concentration of lactic acid in the CFS was measured
to assess its potential effects on antimicrobial activity and cell viability
[2]. The quantity of each form of the acid in the sample was
measured using a commercially available D-lactic
acid/L-lactic acid kit (Roche
Boehringer, Mannheim, Germany), according to the
manufacturer’s instructions.


## 2.4. EpiVaginal Ectocervical Tissue Model

The
EpiVaginal (VEC-100) ectocervical tissue model (MatTek Corporation, Ashland, Mass, USA) was used and maintained as
fully described by Dover et al. [29]. The tissues were exposed to 83μL of subtilosin CFS (∼136μg/mL) for 4, 24, and 48 hours. For exposure times over 24 hours, the
tissues were aerated by placing them on two metal washers (MatTek Corporation, Ashland, Mass, USA) and fed with 5 mL of the assay
medium. Double-distilled water (ddH2O) was used as a negative
control, and was applied to cells after 6, 24, and 48 hours. A spermicidal
product containing 4% Nonoxynol-9 (Ortho Options CONCEPTROL Vaginal
Contraception Gel, Advanced Care Products, Skillman, NJ, USA) was used as a positive control
based on its documented cytotoxic properties [30, 31]. A cream (Monistat-3, Ortho
McNeil  Pharmaceutical, Inc., Raritan, NJ, USA) containing 4% of the nontoxic,
BV-active compound miconazole nitrate [30, 31], was used as a negative control.Following the designated exposure
times, the MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide) assay was used to determine overall cell viability. The data were used
to approximate an effective time (ET) that would reduce cell viability to 50%
(ET-50).


## 2.5. MTT Viability Assay

The
MTT assay was carried out according to the protocol outlined by Dover et al. [29].
Briefly, the viability of ectocervical cells after exposure to subtilosin was
measured as a direct proportion of the breakdown of the yellow compound
tetrazolium to the purple compound formazan, since only living cells can cause
this reaction to occur [32]. Tissues were exposed to subtilosin and
the two controls for several designated time points; at the conclusion of each,
the liquid in the plate wells was combined with the liquid from the tissue
inserts. This mixture was then assayed spectrophotometrically using a 96 well-plate
reader (MRX revelation, Dynex Technologies, Va, USA) to determine the level of
tetrazolium degradation.The viability (%) of the treated
tissue inserts was calculated according to an equation provided by the
manufacturer:%viability=OD570 (treated tissue)/OD570 (negative control tissue). The exposure time that reduced tissue viability by
50% (ET-50) was calculated according to [V=a+b*log(t)]
described by Ayehunie et al. [30], where V=%viability, t=time in minutes, and “a” and “b” are constants representing the viability data from the time points
preceding and following 50% viability. On the whole, there is a direct
relationship between the length of the ET-50 and the toxicity of the tested
application (i.e., a shorter ET-50 corresponds to a more harmful compound).


## 2.6. Semen Sample Collection and Analysis

The
CFS gathered from aB. amyloliquefaciens culture was used to test the effect of subtilosin exposure on the motility of
human spermatozoa. Initially, the CFS was diluted with normal saline (0.9%) so
that 200μL of the final material was equivalent to 50μL, 100μL, or 200μL of undiluted CFS.Two semen samples were collected on
the day of experimentation. Each sample was collected by self-masturbation in a
polypropylene specimen container (Fisher) prior to transport to the laboratory.
Within 1 hour of collection, the samples were pooled. Total sperm count was
calculated using bright field light microscopy (Olympus BX50; 400x) after
dilution (1:50) of the semen in normal saline. The initial percentage of motile
sperm was calculated prior to testing with a neubauer hemacytometer [33]. The determination of motile sperm % was made using randomly
selected field views (400x) from a count of between 104–201 cells. Any
visibly moving spermatozoa (directional or stationary) were counted as  motile cells.The percentage of forward
progressing spermatozoa was subjectively determined based on the assumption
that 70% of the sperm in a normal sample would behave in such a manner. The
samples used in this experimentation fell into such a “normal” category.


## 2.7. Treatment of Spermatozoa with Subtilosin

A modified
Sander-Cramer test was used to determine the effect of column-purified subtilosin
on human spermatozoa motility [34]. This measured the effect
of subtilosin after 30-second exposure times of 5 volumes (200μL) of the solution at each dilution (25% and 50% in
normal saline, and 100%) with one volume (40μL) of whole semen. The motilities of cells from random high-magnification
fields (400x) of the sample were determined in duplicate as described above.


## 2.8. Data Analysis

The % motility data were arcsine transformed
[35] prior to further examination. StatMost32 (version 4.1)
statistical software (DataMost Corporation, Sandy, Utah, USA) was used to calculate all
statistical parameters. The % values of motility were presented as averages and
90% confidence limits. Any differences between treatment groups were assessed
by the Newman-Keuls multiple range test. Differences were deemed significant at
the 0.05 level of confidence.


## 3. Results

### 3.1. Determination of Protein Concentration

The
concentration of subtilosin in the column-purified sample was estimated at135.7μg/mL. The CFS and column-purified sample produced
identical zones of inhibition at each dilution (data not shown); therefore, the
concentrations of protein in both solutions were assumed to be equivalent.
While it is improbable that a 100% yield would be attained from column
chromatography, previous work has shown that protein concentrations can be
precisely calculated based on the comparisons we conducted [36].
Due to the difficulty in measuring the CFS protein concentration via other
assays, the chosen method was deemed the most accurate and reproducible.


### 3.2. Cell Viability% and ET-50 Values

After
48 hours of exposure to subtilosin, the EpiVaginal ectocervical tissues
retained a high level of viability compared to the positive control,
Nonoxynol-9, and the negative control, miconazole nitrate (Table1). Due to the
lack of toxicity of the antimicrobial, the ET-50 value for subtilosin could not
be established since the total cell viability did not drop below 50% at any of
the given time points. However, a projection of the ET-50 value is possible by an
extrapolation of the data. Data presented in Table 1 can be fit to a curve
described by Ln(V)=a+bt2, where a=4.605995356 and b=−0.00014151 (coefficient of determination, or r2, =0.9998), from
which the ET-50 is estimated at 70 hours.Table 1
Ectocervical cell viability after prolonged exposure to subtilosin.
Postexposure cell viability (%)Exposure time (hrs)Assay #SubtilosinNonoxynol-9 (4%)Miconazole nitrate (4%)ddH2O4199.114.1ND100210016.5ND10024194.8058.4100289.8058.310048173.4079.3100270.9083.5100


### 3.3. Quantitative Observations of Motile Spermatozoa

Subtilosin
reduces human sperm motility in a dose-dependent manner (Figure1). The
motility of the treated spermatozoa ranged from 0 to 88% of control motility
levels over the four-fold range of subtilosin concentrations. All of the
subtilosin concentrations tested reduced motility compared to the control
samples. The differences in the proportion of motile spermatozoa in all samples
(28.3, 56.7, and 113.3μg/mL
protein equivalents) were found to be significant (P<.05) according
to the Newman-Keuls multiple range test. TableCurve 2D (ver 5.0) curve-fitting
software (SPSS Scientific Software, Chicago, Ill, USA) was used to fit the data
to a dose-response curve described by Ln (%Motility)=a+b[SubtilosinA]3, where a=4.20781; b=−2.5814e−06; and [subtilosin
A] is expressed as μg/mL
protein equivalents. The curve had a coefficient of determination (r2)=0.9959. The IC50 value,
or the amount of subtilosin required to reduce the motility of spermatozoa in
whole semen by 50%, was calculated to be 64.5μg/mL.Figure 1
Subtilosin A immobilizes human spermatozoa in a
dose-dependent manner. The percentage of motile
spermatozoa in pooled whole semen was determined 30 seconds after mixing with
subtilosin A, at different final concentrations, as indicated. All data were
adjusted to a normal control motility of 70% and subjected to arcsine
transformation before further analysis. Values are expressed as average %
motility. Error bars are 90% confidence limits.


### 3.4. Semiquantitative Observations of Spermatozoa: Forward Progression

Similar
to motility, forward progression of spermatozoa is reduced in a dose-dependent
fashion by subtilosin. In control samples, 70% of sperm exhibited forward
progression; in the presence of50μL
subtilosin this decreased to 50–70%, while 100μL caused a decline to only 10% forward progression. All forward
progression was eliminated after treatment with 200μL subtilosin, with most sperm tails becoming coiled.


## 3.1. Determination of Protein Concentration

The
concentration of subtilosin in the column-purified sample was estimated at135.7μg/mL. The CFS and column-purified sample produced
identical zones of inhibition at each dilution (data not shown); therefore, the
concentrations of protein in both solutions were assumed to be equivalent.
While it is improbable that a 100% yield would be attained from column
chromatography, previous work has shown that protein concentrations can be
precisely calculated based on the comparisons we conducted [36].
Due to the difficulty in measuring the CFS protein concentration via other
assays, the chosen method was deemed the most accurate and reproducible.


## 3.2. Cell Viability% and ET-50 Values

After
48 hours of exposure to subtilosin, the EpiVaginal ectocervical tissues
retained a high level of viability compared to the positive control,
Nonoxynol-9, and the negative control, miconazole nitrate (Table1). Due to the
lack of toxicity of the antimicrobial, the ET-50 value for subtilosin could not
be established since the total cell viability did not drop below 50% at any of
the given time points. However, a projection of the ET-50 value is possible by an
extrapolation of the data. Data presented in Table 1 can be fit to a curve
described by Ln(V)=a+bt2, where a=4.605995356 and b=−0.00014151 (coefficient of determination, or r2, =0.9998), from
which the ET-50 is estimated at 70 hours.Table 1
Ectocervical cell viability after prolonged exposure to subtilosin.
Postexposure cell viability (%)Exposure time (hrs)Assay #SubtilosinNonoxynol-9 (4%)Miconazole nitrate (4%)ddH2O4199.114.1ND100210016.5ND10024194.8058.4100289.8058.310048173.4079.3100270.9083.5100


## 3.3. Quantitative Observations of Motile Spermatozoa

Subtilosin
reduces human sperm motility in a dose-dependent manner (Figure1). The
motility of the treated spermatozoa ranged from 0 to 88% of control motility
levels over the four-fold range of subtilosin concentrations. All of the
subtilosin concentrations tested reduced motility compared to the control
samples. The differences in the proportion of motile spermatozoa in all samples
(28.3, 56.7, and 113.3μg/mL
protein equivalents) were found to be significant (P<.05) according
to the Newman-Keuls multiple range test. TableCurve 2D (ver 5.0) curve-fitting
software (SPSS Scientific Software, Chicago, Ill, USA) was used to fit the data
to a dose-response curve described by Ln (%Motility)=a+b[SubtilosinA]3, where a=4.20781; b=−2.5814e−06; and [subtilosin
A] is expressed as μg/mL
protein equivalents. The curve had a coefficient of determination (r2)=0.9959. The IC50 value,
or the amount of subtilosin required to reduce the motility of spermatozoa in
whole semen by 50%, was calculated to be 64.5μg/mL.Figure 1
Subtilosin A immobilizes human spermatozoa in a
dose-dependent manner. The percentage of motile
spermatozoa in pooled whole semen was determined 30 seconds after mixing with
subtilosin A, at different final concentrations, as indicated. All data were
adjusted to a normal control motility of 70% and subjected to arcsine
transformation before further analysis. Values are expressed as average %
motility. Error bars are 90% confidence limits.


## 3.4. Semiquantitative Observations of Spermatozoa: Forward Progression

Similar
to motility, forward progression of spermatozoa is reduced in a dose-dependent
fashion by subtilosin. In control samples, 70% of sperm exhibited forward
progression; in the presence of50μL
subtilosin this decreased to 50–70%, while 100μL caused a decline to only 10% forward progression. All forward
progression was eliminated after treatment with 200μL subtilosin, with most sperm tails becoming coiled.


## 4. Discussion

TheB. amyloliquefaciens-produced
bacteriocin subtilosin has proven antimicrobial activity against the vaginal
pathogen G. vaginalis, but was not
harmful to the normal and healthy Lactobacillus vaginal microbiota. Data from human vaginal cell viability assays convincingly
demonstrated the safety of subtilosin for human applications in comparison to
other accepted and available products, indicating it could be safely
incorporated into personal care applications aimed at the treatment of
bacterial vaginosis. Prior research in our laboratory that involved similar
studies with the EpiVaginal model was also carried out in conjunction with in vivo testing of the rabbit vaginal
irritation (RVI) system, which doubly confirmed the safety of another
antimicrobial peptide, Lactocin 160 [29]. Therefore, we are confident
that using the EpiVaginal model instead of animal testing to demonstrate the
safety of subtilosin has provided reliable and valid results.Subtilosin was also found to significantly reduce the
motility of human spermatozoa in a concentration-dependent manner for all
concentrations tested.  The effect of
subtilosin on the forward progression of spermatozoa was also observed to be a
dose-dependent interaction. Serial dilutions showed a steady decline in forward
progression, with all progression halted at the highest concentration tested.
It was also noted that at the highest concentration, the tails of the sperm
cells were curved or coiled, indicating the cells were damaged beyond a simple
restriction of movement. Coiling of the cells is considered to be a sperm
abnormality, and may indicate damage to the plasma membrane [37]. Tail coiling has been observed after in vitro exposure of monkey spermatozoa to methyl mercury
[38]. These results suggest that subtilosin can be
established as a general spermicidal agent. It is worth noting that nisin, a
bacteriocin given generally recognized as safe (GRAS) status by the Food and Drug
Administration (FDA), was also shown to have impressive spermicidal activity
[25]. However, the allure of these results is diminished due to
the fact that it also has potent antimicrobial activity against healthy vaginal
microbiota (Chikindas et al., unpublished data), a strong detraction from its
commercial applicability. Therefore, subtilosin’s spermicidal activity, combined
with its overall safety to both human tissues and healthy human microbiota, make
it a highly recommendable compound for inclusion in topical BV treatments and
human contraceptive products. To facilitate the process of its incorporation
into contraceptive treatments, additional analysis will be done to determine
the reversibility of damage done to the spermatozoa, as well as a time course
assay to further elucidate the exact changes effected by subtilosin.



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*Source: 540758-2008-10-09.xml*