Vaginal Lactobacilli Reduce Neisseria gonorrhoeae Viability through Multiple Strategies: An in Vitro Study

Abstract

The emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae (GC) underline the need of "antibiotic-free" strategies for the control of gonorrhea. The aim of this study was to assess the anti-gonococcal activity of 14 vaginal Lactobacillus strains, belonging to different species (L. crispatus, L. gasseri, L. vaginalis), isolated from healthy pre-menopausal women. In particular, we performed "inhibition" experiments, evaluating the ability of both lactobacilli cells and culture supernatants in reducing GC viability, at two different contact times (7 and 60 min). First, we found that the acidic environment, associated to lactobacilli metabolism, is extremely effective in counteracting GC growth, in a pH- and time-dependent manner. Indeed, a complete abolishment of GC viability by lactobacilli supernatants was observed only for pH values < 4.0, even at short contact times. On the contrary, for higher pH values, no 100%-reduction of GC growth was reached at any contact time. Experiments with organic/inorganic acid solutions confirmed the strict correlation between the pH levels and the anti-gonococcal effect. In this context, the presence of lactate seemed to be crucial for the anti-gonococcal activity, especially for pH values in the range 4.4-5.3, indicating that the presence of H⁺ ions is necessary but not sufficient to kill gonococci. Moreover, experiments with buffered supernatants led to exclude a direct role in the GC killing by other bioactive molecules produced by lactobacilli. Second, we noticed that lactobacilli cells are able to reduce GC viability and to co-aggregate with gonococci. In this context, we demonstrated that released-surface components with biosurfactant properties, isolated from "highly-aggregating" lactobacilli, could affect GC viability. The antimicrobial potential of biosurfactants isolated from lactobacilli against pathogens has been largely investigated, but this is the first report about a possible use of these molecules in order to counteract GC infectivity. In conclusion, we identified specific Lactobacillus strains, mainly belonging to L. crispatus species, able to counteract GC viability through multiple mechanisms. These L. crispatus strains could represent a new potential probiotic strategy for the prevention of GC infections in women.

Keywords: Lactobacillus; STIs; gonococcus; gonorrhea; probiotics.

Figure 1

“Inhibition” experiments with lactobacilli CFS. Experiments were performed with a volume of supernatants corresponding to 1 × 10⁸ CFU of lactobacilli added to 1 × 10⁸ gonococcal cells at two different time points: 7 min (dotted bars) and 60 min (gray bars). GC viability was evaluated as number of gonococci/mL and expressed as % of gonococci survival. The results were expressed in percentage compared to control (K) (1 × 10⁸ gonococcal cells incubated in MRS), taken as 100% (black bar). Bars represent mean values, whereas error bars represent SEM. Statistical significance was determined at ^**P < 0.01 and ^***P < 0.0001.

Figure 2

Effect of lactic and hydrochloric acid on GC viability. Solutions of hydrochloric acid (HCl) and lactic acid (LA) at a concentration of 10 mM, buffered to different pH values (3.4, 3.7, 4.0, 4.4, 4.7, 5.0, 5.3), were used for “inhibition” experiments against GC at two different time points (7 and 60 min; dotted and gray bars, respectively). GC viability was evaluated as number of gonococci/mL and expressed as % of gonococci survival. The results were expressed in percentage compared with control (K) (1 × 10⁸ gonococcal cells incubated in 150 mM NaCl solution, pH 5.8), taken as 100% (black bar). Bars represent mean values, whereas error bars represent SEM. Statistical significance was determined at ^*P < 0.05, ^**P < 0.01, and ^***P < 0.0001.

Figure 3

“Inhibition” experiments with lactobacilli cells. Experiments were performed with 1 × 10⁸ CFU of lactobacilli incubated with 1 × 10⁸ gonococcal cells at two different time points: 7 min (dotted bars) and 60 min (gray bars). GC viability was evaluated as number of gonococci/mL and expressed as % of gonococci survival. The results were expressed in percentage compared to control (K) (1 × 10⁸ gonococcal cells incubated in PBS, without lactobacilli), taken as 100% (black bar). Bars represent mean values, whereas error bars represent SEM. Statistical significance was determined at ^*P < 0.05, ^**P < 0.01, and ^***P < 0.0001.

Figure 4

Effect of lactobacilli RSC on GC viability. “Inhibition” experiments against GC were performed with released surface components isolated from L. crispatus BC1 (BC1) and L. gasseri BC10 (BC10). Three different dilutions of RSC were tested (1:1, 1:2, and 1:4) at two different time points (7 min-dotted bars and 60 min-gray bars). GC viability was evaluated as number of gonococci/mL and expressed as % of gonococci survival. The results were expressed in percentage compared to control (K) (1 × 10⁸ gonococcal cells incubated in PBS), taken as 100% (black bar). Bars represent mean values, whereas error bars represent SEM. Statistical significance was determined at ^*P < 0.05, ^**P < 0.01, and ^***P < 0.0001.

Figure 5

Lactobacilli interaction with piliated GC. On the left: L. crispatus BC1 shows high levels of interaction with GC after 7 min contact time, as underlined by the formation of lactobacilli-gonococci cells aggregates. On the right: after an interaction of 7 min, the ability of L. crispatus BC6 to aggregate with gonococci was excluded. Gram-stained images at 1,000 × magnification.