Developing and Applying Microdroplet Co-Cultivation Technology for Elucidating Bacterial Interspecies Interactions in the Human Vaginal Microbiome

Abstract

The role of the human vaginal microbiome (HVM) has gained increased recognition due to recent technological advancements that helped link community composition and women's health risks. However, the ecological roles of members of the HVM and microbe-microbe-host interactions remain unclear. Current approaches for investigating these mechanisms have been low-throughput, require large cultivation volumes and utilize chemically indistinct media diverging from the in vivo condition. Microdroplet-based co-cultivation is a new technology for overcoming these challenges by confining and performing sensitive assays at the nano-liter scale. This dissertation aims to: (i) develop new methods for elucidating interactions in the HVM through co-cultivation in microdroplets; (ii) test hypotheses that reduced iron limits the growth of L. iners and study interactions between lactobacilli in co-culture in laboratory media; and (iii) further extend the microdroplet technology for culturing vaginal bacteria in pooled cervicovaginal fluid (CVF).

First, we adapted and extended a microdroplet co-cultivation technology pipeline to investigate the HVM and tested it using two pairwise model systems. In one case, Lactobacillus jensenii JV-V16, a lactic-acid bacterium, and Gardnerella vaginalis ATCC 49145, a putative pathogen, were cultured in microdroplets as pure cultures and co-cultures. Then, qPCR was used to quantify the bacteria in pooled microdroplets, and individual microdroplets were isolated and cells within each were plated on agar media. We demonstrated that L. jensenii inhibits G. vaginalis in microdroplets, which concurs with flask cultivation studies. We further demonstrated a second model system consisting of L. jensenii and another potential pathogen, Enterococcus faecalis. Our findings suggest that microdroplets can detect microbial interactions.

Second, we determined the effects of iron on the growth of the most common lactobacilli, L. iners and L. crispatus, and investigated pairwise interactions between lactobacilli using laboratory media. We measured the growth of L. iners and L. crispatus in spent-media supplemented with Fe(II)SO4 or 2,2'-dipyridyl. Results show that higher concentrations of 2,2'-dipyridyl reduced the growth of L. iners, but not that of L. crispatus. We conducted serial dilutions on co-cultures of L. crispatus and L. iners, and L. crispatus and L. gasseri. As observed, one species became the most dominant in each co-culture. Spent-medium experiments indicated that no interference competition existed between these lactobacilli. Future investigation is needed to identify mechanisms for resource competition.

Third, we extended our technology to cultivate vaginal bacteria in microdroplets using CVF and investigated whether L. crispatus, L. gasseri, or L. iners could grow in pooled CVF. We analyzed 16S rRNA genes of 49 vaginal samples collected from healthy reproductive-age women. Of them, 16 were selectively pooled to create L. crispatus (LC)-dominated CVF. Using microdroplets, we subsequently confined and axenically cultured L. crispatus, L. iners, and L. gasseri in LC-CVF. We observed that L. iners grew in LC-CVF at pH 7 but was killed at pH 4. Our results indicate how vaginal pH may influence L. iners growth. L. crispatus survived and L. gasseri decreased in viability in LC-CVF at pH 4.

In conclusion, this dissertation demonstrates methods for investigating interactions in the HVM through co-cultivation in microdroplets in a high-throughput manner. We have also shown the utilization of small volumes of human samples in cultivating vaginal bacteria while simulating the natural condition of the vagina. Further extension of this approach and its future applications hold tremendous potential for elucidating microbial interactions and how they impact human health.