To successfully infect the female genital tract, gonococci must first adhere to the epithelia to avoid clearance by the continual flow of vaginal fluid. A reduction in the ability to adhere would significantly affect the outcome of an infection following exposure. Although lactobacilli have been implicated in protecting the female vaginal tract from exogenous pathogens, research on the direct effect of lactobacilli on STIs has been lacking. Therefore, we developed a coculture model of gonococcal infection that includes lactobacilli to examine their effect on gonococcal interactions with epithelial cells.
In our in vitro model of gonococcal infection, precolonization with lactobacilli reduced gonococcal adherence to epithelial cells by 40 to 50% (Fig. and ). This inhibition of adherence was dependent on the ratio of gonococci to adherent lactobacilli, with the inhibition becoming more pronounced in the presence of increased numbers of lactobacilli (Fig. ). In a gonococcal infection, following adherence to epithelial cells, gonococci will invade these epithelial cells and transcytose to the subepithelial space. Our results show that gonococcal invasion of epithelial cells precolonized with L. jensenii was decreased by 60%. This inhibition of invasion was distinct from the inhibition of adherence, demonstrating that even the gonococci that overcome the inhibition of adherence are negatively affected in invasiveness by the presence of lactobacilli. The inhibition of adherence, along with the inhibition of invasion, suggests that colonization with lactobacilli could reduce the risk for infection by N. gonorrhoeae by potentially decreasing the amount of gonococci that adhere and invade the mucosal epithelia, thus providing a greater opportunity for the innate host defenses to prevent the establishment of an infection.
Due to the nature of N. gonorrhoeae
as an obligate human pathogen, testing the biological relevance of the 50% reduction in gonococcal adherence caused by lactobacilli cannot be done in vivo. However, semen samples from males infected with gonorrhea on average contain 7 × 106
CFU per ejaculate (12
), similar to the gonococcal inoculum used in these studies (6 × 106
CFU/well). Cervical aspirates of females with gonorrhea have been reported to contain 104
). In our studies the average number of CFU of gonococci adherent to epithelial cells in the controls falls within this range (1 × 106
CFU/well), again lending support to the relevance of our experimental conditions. Thus, the inhibition of gonococcal adherence to and invasion of epithelial cells precolonized with lactobacilli may explain the epidemiological observations in which women with bacterial vaginosis (reduced numbers of lactobacilli) have an increased susceptibility for gonorrhea following exposure (32
Lactobacilli are now being promoted as probiotic bacteria that have broad health benefits. Lactobacilli have the ability to displace adherent pathogens associated with urinary tract infections (23
), as well as Gardnerella vaginalis
biofilms associated with BV (25
), suggesting potential as a treatment for these diseases. While it is unlikely that lactobacilli alone could be used as a treatment for gonorrhea, it is conceivable that lactobacilli could reduce the likelihood that gonococci would establish an infection when lactobacilli are directly administered to the vaginal tract shortly after exposure. Reid et al. (23
) suggests a displacement of 20 to 50% would be clinically relevant for pathogens infecting the vaginal tract. Our results indicate that gonococcal displacement by L. gasseri
ATCC 33323 is within this range at 28.7% (Fig. ). Thus, the ability of lactobacilli to displace adherent gonococci in a cell culture model of infection suggests that lactobacilli have potential as a postexposure prophylactic for gonococcal infection.
In this study, L. jensenii
and L. gasseri
strains were chosen because these species are two of the most prevalent species recovered from healthy women and because they had been previously studied for their effects on N. gonorrhoeae
in the absence of epithelial cells (1
). Each strain inhibited the adherence of N. gonorrhoeae
to epithelial cells but adhered poorly to epithelial cells (average adherence frequency of ~1%). When epithelial cells are precolonized with lactobacilli at an MOI of 100, there is on average 1 adherent Lactobacillus
per epithelial cell. Thus, it is unlikely that the mechanism for exclusion of gonococci is direct competition for receptors on the epithelial cells. However, the number of adherent lactobacilli is inversely correlated with the gonococcal adherence frequency: as the number of adherent lactobacilli increases, the gonococcal adherence frequency decreases. This suggests that it is the adherent lactobacilli that play an important role in the observed inhibition of gonococcal adherence. Our results also show that the mechanism of inhibition of adherence is not due to coaggregation of lactobacilli with gonococci (Fig. ), growth inhibition (Fig. ), or the secretion of a stable soluble compound into the medium by either the epithelial cells or lactobacilli before inoculation with gonococci (Fig. ). Further study will be necessary to identify the mechanisms of inhibition, which will be fundamental in the development of Lactobacillus
-based treatments or preventatives for gonococcal infection.
In summary, we have adapted a tissue culture model of gonococcal infection to include a major constituent of the human vaginal tract, lactobacilli. This approach will be essential in elucidating the role of the indigenous microbiota in the early, and therefore preventable, steps of a gonococcal infection. The observation that lactobacilli inhibit gonococcal adherence and invasion and could even displace adherent gonococci is intriguing; however, the mechanisms by which these events occur are not yet clear. While coaggregation, the secretion of soluble inhibitory molecules, and growth inhibition can be ruled out as possible mechanisms, there are still several hypotheses to test. Possible mechanisms might include a contact-inducible change in the epithelial cells due to the adherence of lactobacilli that would reduce susceptibility to gonococci or the secretion of a short-lived inhibitory factor produced by lactobacilli directly in response to incoming gonococci. This model will be indispensable in future studies to examine the molecular basis for the observed inhibition of gonococcal interactions with epithelial cells.