bacterial   vaginosis ( bv ) is the most frequent   vaginal 
 infectious disorder in women of childbearing age with prevalences ranging from 
 10% to 50% . in addition to the physiological burden that induces bv , it can cause 
 serious sequelae such as preterm birth and facilitate the acquisition of 
 sexually transmitted diseases . the cause of bv remains poorly understood , and 
 no specific infectious agents have been identified . however , the disorder is 
 characterized by modifications of the genital tract microflora , including a 
 reduction in or absence of lactobacillus colonization and overgrowth of several 
 anaerobic bacteria . the vaginal ecosystem in healthy premenopausal women harbors a 
 microbiota dominated by lactobacilli [ 3 , 4 ] , that is , being increasingly recognized as protecting it from invading 
 pathogens , including those that cause urinary tract infections and sexually 
 transmitted diseases . different mechanisms are potentially involved in the 
 activity of lactobacilli against 
 pathogens , including the competitive exclusion of genitourinary pathogens from 
 receptors present on the surface of the epithelial cells . under healthy 
 conditions , the recommended treatment regimens for vaginal infections 
 are oral or intravaginal antibiotics , but these conventional treatments are associated with frequent 
 recurrences . alternative therapeutic agents need to be sought , and it has been 
 suggested that the administration of lactobacilli can restore ecological balance in the vagina by controlling the infectivity of 
 pathogenic microbes , but the treatment is still a subject of debate . several clinical 
 trials have been performed to investigate the effects of specific strains , 
 mainly with l. acidophilus and l. rhamnosus species [ 6 , 7 ] , but no definitive conclusions as to whether these probiotics represent 
 an effective and safe method for treating women with bv can be drawn . the 
 behavior of the probiotics in the vaginal tract is likely to be strain specific 
 and therefore , it is important to determine the characteristics of the strain 
 to be used as a therapeutic agent . the most relevant properties in this context 
 are likely to be adhesion to cervicovaginal cells and adequate pathogen growth 
 inhibition . in vitro studies assessing these properties might not be able to 
 fully simulate the in vivo behavior , but they could be reliable indicators when 
 selecting the probiotic strain . the purpose of this study was to determine the 
 in vitro adherence of a well characterized l. 
 rhamnosus probiotic strain , lcr35 [ 810 ] , and its ability to inhibit growth of three vaginosis - associated 
 pathogens . we used immortalized morphologically and functionally distinct 
 epithelial cell lines from normal endocervix , ectocervix , and vagina to 
 characterize lcr35 epithelial interactions pertinent to the lower female 
 genital tract and determined its antimicrobial activity against prevotella bivia , gardnerella vaginalis , and candida 
 albicans in coculture experiments . adhesion assays were performed with epithelial cells from 
 normal human vagina ( vk2/e6e7 atcc - crl-2616 ) , ectocervix ( ect1/e6e7 
 atcc - crl-2614 ) , and endocervix ( end1/e6e7 atcc - crl-2615 ) , immortalized by 
 expression of the e6 and e7 genes of human papillomavirus type 16 . the morphological and immunocytochemical characteristics of the 
 immortalized lines closely resembled those of their tissues of origin and 
 primary cultures and are likely to represent the different compartments of the 
 vaginal tract . the cell lines were maintained in keratinocyte serum - free 
 medium ( gifco brl 17005 - 042 ) supplemented with human recombinant egf ( 0.1 ng / ml ) , 
 bovine pituitary extract ( 0.05 mg / ml ) , and calcium chloride ( 0.4 mm ) at 37c 
 with a 5%   co2 in air atmosphere . adhesion of the lcr35 was assayed by seeding cell lines in 
 24-well tissue culture plates at 2.5  10 epithelial cells / well and 
 allowing them to grow to complete confluence ( 10 cells / well ) . after gentle washing of the cell monolayer , 
 the adhesion capacity of lcr35 was determined by adding 10 multiplicity of infection 
 ( moi,1 ) , 10 ( moi , 10 ) , and 10 ( moi , 100 ) bacteria from 
 an overnight culture in de man , rogosa , sharpe ( mrs ) agar medium . bacterial cells were previously washed in 
 phosphate buffered saline and resuspended in the cell culture medium . adhesion 
 was monitored after 1 and 3 hours of incubation carried out at 37c under 5%   co2 . the monolayers were washed three times with 1 ml of dulbecco 's phosphate 
 buffered saline , detached by addition of 0.1% tritonx-100 solution and the number 
 of viable bacteria determined by plating serial dilutions of the suspensions 
 onto mrs agar plates . for qualitative analysis , the cell monolayers and the 
 bacteria were methanol fixed and stained by addition of a 10% giemsa solution . the effect of lcr35 on the growth of three pathogens was 
 investigated using the following strains : candida albicans atcc10231 , prevotella bivia atcc29303 , and gardnerella vaginalis atcc14018 . coculture assays were performed in either sabouraud broth ( candida ) or brain 
 heart infusion supplemented with yeast extract ( 1% ) , maltose ( 0.1% ) , glucose 
 ( 0.1% ) , and horse serum ( 10% ) ( prevotella and gardnerella ) . each pathogen ( 10 ufc ) was incubated alone ( control ) and with the lcr35 ( 10 ufc ) at 
 37c under anaerobic conditions for the two vaginosis - associated bacteria 
 ( anaerogen , oxod ) . aliquots were removed after 4 , 8 , and 24 hours of 
 incubation , serially diluted and plated on appropriate media ( sabouraud , 
 gardnerella , or mrs ) to determine the bacterial colony counts of both the 
 pathogens and lcr35 . the ability of lcr35 to adhere to vaginal and cervical 
 cells is shown in figure 1 . whatever the moi and the cell line , the probiotic 
 strain was able to adhere to the cell surface monolayer . the highest number of 
 adherent bacteria was observed with the vaginal cell line , with an average of 
 4.75 10e5 cfu per cm after 1 hour of incubation . no major 
 difference was observed between the levels of adhesion obtained after 1 hour 
 and 3 hours of incubation ( data not shown ) , suggesting that adhesion occurs 
 rapidly after the initial contact between the cells and the bacteria . microscopical observations of giemsa - stained preparation showed typical chains 
 of lcr35 randomly dispersed on the cell surface ( figure 2 ) . the antagonist effect of lcr35 against three main 
 pathogens , p. bivia , g. vaginalis , and c. albicans , was assessed in coculture assays and compared with the 
 growth ability of each pathogen in the same culture medium ( figure 3 ) . a 
 decrease in the cell division rate of the three microorganisms tested was 
 observed from 4 hours of coincubation . when the viable bacteria in the mixed 
 suspension were counted over a longer period of time , bactericidal activity was 
 detected between 8 and 24 hours of incubation for all pathogens with the prevotella strain being the most 
 susceptible ( 4-log10 units decrease in the number of viable cells ) . in no case there was a bactericidal effect against lactobacilli ; 
 the number of viable lcr35 cells was either constant over the incubation period 
 ( coculture with c. albicans ) or 
 increased ( coculture with g. vaginalis and p. bivia ) ( data not shown ) . lactobacillus species in the female urogenital system act as a barrier 
 to infection and   contribute to the control of the vaginal microbiota by 
 competing with other microorganisms for adherence to epithelial cells , 
 displacing pathogen biofilm [ 12 , 13 ] , and/or inhibiting the growth of potential pathogens [ 1416 ] . hence the use of probiotic strains of lactobacilli is potentially interesting both as preventive and 
 curative agents . unlike the use of vaginal epithelial cells 
 collected from healthy premenopausal women , assays 
 performed   with immortalized   epithelial cell lines , which closely resemble the 
 epithelial differentiation patterns of normal human tissues , are more accurate 
 for standardizing tested bacterial adherence and allow comparison of different 
 research approaches . the three epithelial cell lines tested in this study were 
 developed from normal human vagina , ectocervix , and endocervix tissue , and 
 their characteristics closely resembled those of their tissues of origin and 
 primary cultures . we can thus speculate that adhesion assays performed with this 
 material reproduce more faithfully the in vivo situation than experiments 
 performed with any cell line derived from human carcinoma of the lower genital 
 tract mucosa . this is particularly 
 important when comparing bacterial strains belonging to the complex lactobacillus genus that includes 
 bacterial strains with highly specific characteristics . using these cell lines , 
 we observed specific adhesion of an l. 
 rhamnosus strain , lcr35 , previously selected for its probiotic features [ 810 ] . adhesion occurred even at a low moi ( 1:1 ) and within less than 1 hour 
 of contact , which corresponds to a highly dynamic process . adhesion of lcr35 to vaginal epithelial cells 
 would allow colonization of the vaginal mucosa and therefore could limit the 
 overgrowth of pathogens , but the second main property of a potential probiotic 
 used as a therapeutic agent against pathogenic microorganisms is direct impairment 
 of their growth . in this study , we demonstrated that lcr35 showed bactericidal 
 activity against both p. bivia and g. vaginalis in the range of killing 
 stipulated for the bactericidal activity of antimicrobial activity ( > 2 
 log - unit ) . in a previous study , atassi et al . demonstrated that the 
 bactericidal activity of lactobacilli toward these two vaginal bacterial pathogens was strain dependent and occurred 
 within the first hours of coculture . in our experiments , a longer incubation time was required to observe 
 bactericidal activity , probably because of the different experimental 
 parameters used in the two assays . we previously showed that the lcr35 
 probiotic strain was also able to kill several pathogens . the mechanism(s ) underlying this activity has not been elucidated but 
 is likely to be multifaceted and probably includes the production of hydrogen 
 peroxide , lactic acid , and antibacterial compounds . it has been recently shown 
 that g. vaginalis organized in 
 biofilms is more 
 resistant to h2o2 and lactic acid than planktonic 
 cultures . g. vaginalis is the 
 predominant species observed within biofilms present on the vaginal epithelium 
 in bacterial vaginosis , and saunders et al . it would therefore be interesting to test the biofilm activity of 
 lcr35 against g. vaginalis and to 
 determine if the sessile form of lcr35 also exhibits antibacterial activity 
 against g. vaginalis in mixed biofilm 
 assays . the antagonist activity of lcr35 was not limited to 
 bacterial pathogens since the strain was also able to reduce the viability of c. albicans . several strains of lactobacilli have shown inhibitory 
 effects against c. albicans , which is the species most often associated with candidiasis . by 
 interfering with candida overgrowth 
 in the patients ' intestinal or vaginal tract , lactobacilli could provide colonization resistance and maintain low 
 numbers of yeasts , especially when administered together with antibiotics . relevant clinical trials have suggested that 
 intravaginal or oral administration of lactobacillus strains is able to 
 increase the numbers of vaginal lactobacilli and restore the vaginal microbiota to normal . lcr35 was isolated from a human intestinal and not vaginal microbiota 
 and does not belong to the four main species of lactobacilli considered to be predominantly linked to the vaginal 
 microflora , l. crispatus , l. jensenii , l. gasseri , and l. iners [ 4 , 19 , 20 ] , but it has been shown to survive within the human gastrointestinal 
 tract . furthermore , petricevic and witt recently showed in a clinical study 
 that topical   administration of lcr35 enhances the restoration of the vaginal 
 flora after antibiotic treatment of bv . thus , it might be an excellent candidate for use as a prophylactic 
 agent , taken orally or applied topically . in vivo studies to evaluate its 
 feasibility as such are in progress . maintenance or reconstruction of the normal composition of 
 the vaginal microflora by applying properly selected lactobacilli may be of prophylactic value in preventing or curing 
 genitourinary system infections in women . in the light of our experiments , it 
 seems that the probiotic strain l. 
 rhamnosus lcr35 would be a good candidate as a protective agent against 
 both bacterial vaginosis and candida vaginitis since it was able to adhere to vaginal and cervical cells and to 
 antagonist the growth of vaginosis - associated pathogens . clinical studies are 
 now required to assess the in vivo efficacy of such a therapy .
objectives . the ability of a probiotic lactobacillus rhamnosus strain ( lcr35 ) to adhere to cervical and vaginal cells and to affect the viability of two main vaginosis - associated pathogens , prevotella bivia , gardnerella vaginalis , as well as candida albicans was investigated . 
 methods . 
 adhesion ability was determined in vitro with immortalized epithelial cells from the endocervix , ectocervix , and vagina . 
 coculture experiments were performed to count viable pathogens cells in the presence of lcr35 . 
 results . 
 lcr35 was able to specifically and rapidly adhere to the three cell lines . in coculture assays , 
 a decrease in pathogen cell division rate was observed as from 4 hours of incubation and bactericidal activity after a longer period of incubation , mostly with p. bivia . 
 conclusion . 
 the ability of lcr35 to adhere to cervicovaginal cells and its antagonist activities against vaginosis - associated pathogens suggest that this probiotic strain is a promising candidate for use in therapy .