subtilosin 
 is a ribosomally - synthesized   cyclopeptide produced by bacillus subtilis atcc 6633 and   a recently identified natural isolate   of bacillus amyloliquefaciens [ 1 , 2 ] .
this protein has a unique structure 
 among bacteriocins , and possesses antimicrobial activity 
 against a variety of pathogenic organisms , including gardnerella vaginalis , listeria 
 monocytogenes , and streptococcus 
 agalactiae ( group b streptococcus ) .
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 .
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 
 [ 57 ] .
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 [ 711 ] . 

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 . 

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 
 [ 1216 ] .  
the treatments recommended by the centers for 
 disease control ( cdc ) are clindamycin and metronidazole administered either 
 orally or intravaginally .   following these guidelines
successfully 
 treats 60% of bv cases , but 20% of these cases return with highly developed antibiotic 
 resistances [ 1820 ] . 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 .
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 .
they are especially appealing alternatives 
 since their cost of production is so comparatively low to conventional 
 pharmaceutical treatments .
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 , has human spermicidal activity [ 24 , 25 ] , and subtilosin has spermicidal activity against boar , bovine , 
 horse , and rat spermatozoa .
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 .
subtilosin 
 was prepared as previously described . 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 4c .
the supernatant was filter sterilized using 0.45 m 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 4c and was resuspended in 
 20 ml of double distilled h2o .
the column chromatography method 
 described by sutyak et al .   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 .   with additional modifications .
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 .
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 of 
 cu to cu by colorimetric detection of cu by bicinchoninic acid . bovine serum albumin ( bsa )
was used to develop a 
 standard curve with concentrations ranging from 0.5 to 20 g / ml ; the concentration of subtilosin was calculated using the r value 
 from the trendline of the standard curve graph .
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 using 50 l 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 .
as 
 reported previously , the concentration of lactic acid in the cfs was measured 
 to assess its potential effects on antimicrobial activity and cell viability 
 .
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 .
the 
 epivaginal ( vec-100 ) ectocervical tissue model ( mattek corporation , ashland , mass , usa ) was used and maintained as 
 fully described by dover et al . .
the tissues were exposed to 83 l of subtilosin cfs ( 136 g / 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 ) .
the 
 mtt assay was carried out according to the protocol outlined by dover et al . . 

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 .
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 . , 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 ) .
the 
 cfs gathered from a b. 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 200 l of the final material was equivalent to 50 l , 100 l , or 200 l of undiluted cfs .
each sample was collected by self - masturbation in a 
 polypropylene specimen container ( fisher ) prior to transport to the laboratory . 
 within 1 hour of collection ,
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 .
the determination of motile sperm % was made using randomly 
 selected field views ( 400x ) from a count of between 104201 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 .
a modified 
 sander - cramer test was used to determine the effect of column - purified subtilosin 
 on human spermatozoa motility .
this measured the effect 
 of subtilosin after 30-second exposure times of 5 volumes ( 200 l ) of the solution at each dilution ( 25% and 50% in 
 normal saline , and 100% ) with one volume ( 40 l ) of whole semen .
the motilities of cells from random high - magnification 
 fields ( 400x ) of the sample were determined in duplicate as described above .
statmost32 ( version 4.1 ) 
 statistical software ( datamost corporation , sandy , utah , usa ) was used to calculate all 
 statistical parameters .
any differences between treatment groups were assessed 
 by the newman - keuls multiple range test .
the 
 concentration of subtilosin in the column - purified sample was estimated at 
 135.7 g / 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 . 
 due to the difficulty in measuring the cfs protein concentration via other 
 assays , the chosen method was deemed the most accurate and reproducible .
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 ( table 1 ) . 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 + bt , where a = 4.605995356 and b =   0.00014151 ( coefficient of determination , or r ,   = 0.9998 ) , from 
 which the et-50 is estimated at 70 hours .
subtilosin 
 reduces human sperm motility in a dose - dependent manner ( figure 1 ) .
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 113.3 g / 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 [ subtilosin   a]3 , where a = 4.20781 ; b =   2.5814e  06 ; and [ subtilosin 
 a ] is expressed as g / ml 
 protein equivalents .
the ic50 value , 
 or the amount of subtilosin required to reduce the motility of spermatozoa in 
 whole semen by 50% , was calculated to be 64.5 g / ml .
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 of 50 l 
 subtilosin this decreased to 5070% , while 100 l caused a decline to only 10% forward progression .
all forward 
 progression was eliminated after treatment with 200 l subtilosin , with most sperm tails becoming coiled .
the b. 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 .
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 .
tail coiling has been observed after in vitro exposure of monkey spermatozoa to methyl mercury 
 .
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 
 .
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 .
 bacterial vaginosis ( bv ) , a condition affecting millions of women each year , is primarily caused by the gram - variable organism gardnerella 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 . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION