Insights into host dependency from a chemically defined medium for the human vaginal bacterium Lactobacillus crispatus

Abstract

A Lactobacillus-rich vaginal microbiome is associated with a reduced risk for sexually transmitted diseases and adverse reproductive health outcomes, with Lactobacillus crispatus identified as particularly beneficial. This study investigated the nutritional requirements of two vaginal isolates, L. crispatus RL09 and RL10, and presents a chemically defined medium (CDM) that supports their growth. This study experimentally validated that L. crispatus relies on exogenous fatty acids, essential B vitamins, including riboflavin (B2), niacin (B3) and pantothenate (B5), and all eighteen amino acids for sustained growth. On one hand, this body of work adds to the physiological understanding of Lactobacilliaceae and lays the groundwork for future quantitative studies on L. crispatus. On the other hand, we have shown that L. crispatus exhibits a high metabolic dependency on its environment. These dependencies underscore the potential sensitivity of L. crispatus to nutrient variations, which may influence its ability to dominate and maintain a healthy vaginal ecosystem.

Keywords: Lactobacillaceae; Lactobacillus crispatus; Chemically defined medium; Nutrient requirements; Vaginal microbiome.

Fig. 1

The effect of single and multiple omission of basal components of the chemically defined medium on the growth of L. crispatus RL10. The X axis depicts the omitted nutrient, with (−) being the complete CDM. Left: the Y axis depicts the increase in optical density at 600 nm (ΔOD600 nm). Right: the Y axis represents the measured pH at the end of the batch (pH_endpoint). Three biological replications are shown (●, ▲ and ■), including their technical replicates. Solely the second propagation at 24 h is shown, results of the first and third are available in the supplementary data (Sup. Figure 1)

Fig. 2

The effect of the single or complete omission of amino acids of the chemically defined medium on the biomass growth and acidification of L. crispatus RL09 and RL10. The X-axis depicts the omitted nutrient, with (−) being the complete CDM. Left: the Y axis depicts the increase in optical density at 600 nm (ΔOD600 nm). Right: the Y-axis represents the measured pH at the end of the batch (pH_endpoint). Two biological replications (● and▲) are shown. Solely the second propagation at 24 h is shown, results of the first and third are available in the supplementary data (Sup. Figure 2)

Fig. 3

The effect of the single or complete omission of nucleotides of the chemically defined medium on the biomass growth and acidification of L. crispatus RL10. The X-axis depicts the omitted nutrient, with (−) being the complete CDM. In bold the purines and in normal font the pyrimidines. Left: the Y axis depicts the increase in optical density at 600 nm (ΔOD600 nm). Right: the Y axis represents the measured pH at the end of the batch (pH_endpoint). Two biological replications (●, ▲ and ■) are shown including their technical replicates. Solely the second propagation at 24 h is shown, results of the first and third are available in the supplementary data (Sup. Figure 3)

Fig. 4

The effect of the single or complete omission or addition (vit. B12) of vitamins/nucleotides of the chemically defined medium on the biomass growth and acidification of L. crispatus RL10. The X axis depicts the omitted nutrient, with (-) being the complete CDM or the addition to the cCDM for vit. B12. Left: the Y axis depicts the increase in optical density at 600 nm (ΔOD600 nm). Right: the Y-axis represents the measured pH at the end of the batch (pH_endpoint). Two biological replications (● and▲) are shown, including their technical replicates. Solely the second propagation at 24 h is shown, results of the first and third are available in the supplementary data (Sup. Figure 4)

Fig. 5

Complete CDM v.s. redefined CDM at the 2nd propagation at 24 h and 48 h of growth with L. crispatus RL09 and RL10. They were grown anaerobically at 37 °C on complete CDM and redefined CDM (X axis), and the optical density at 600 nm was measured at 24 and 48 h (Y axis). The error bars depict the standard deviation of three biological replicates, with each two technical replicates