Our research group focuses on the bacteria involved in female genital tract health and disease. This is particularly important in Africa, which has some of the world’s highest rates of bacterial vaginosis (BV). BV is a polymicrobial dysbiosis that is associated with increased risk of STI acquisition (including HIV), complications during pregnancy and other negative health outcomes. Unfortunately, current standard of care BV treatment options often only provide temporary resolution and relapse is the case for up to 80% of women. As a research group, we are interested in understanding the interactions between optimal bacteria (Lactobacillus crispatus) and BV-associated bacteria (Gardnerella spp., Prevotella spp., Fannyhessea and others).

Probiotics represent an effective, sustainable adjunctive treatment for the maintenance of vaginal health. However, despite there being a range of commercial products available, the majority are beyond the price point of most women living in Africa. Importantly, virtually none of the vaginal probiotics currently marketed contain bacterial isolates of vaginal origin. Moreover, those that do typically only contain a single bacterial strain. There is increasing evidence to suggest that strain consortia, rather than individual isolates are better able to recapitulate healthy microbial ecosystems (‘the whole is greater than the sum of the parts’) and that geography matters when choosing probiotic candidates. For example, probiotics isolated from individuals living in a particular geographic area are better able to inhibit the growth of pathogens isolated from individuals in the same area.

In our quest to identify regionally-relevant, affordable probiotic candidates we have identified women in African populations with longitudinally stable, optimal vaginal microbiotas. We have isolated potential probiotic candidates from these women and are trying to understand their interactions with BV-associated bacteria from women in the same populations. Co-culture experiments are a crucial aspect of this and the Cerillo Co-culture system would allow us to build upon the basic proof of concept experiments we have done thus far (bacterial inhibition soft agar overlay assays, incubation in the presence of spent culture medium, etc).

We propose growing our probiotic candidates in the presence of various African Gardnerella, Prevotella and Fannyhessea spp. isolates in the co-culture system to answer the following questions:

1) What happens in a head-to-head competition assay? – Experiment: Inoculate L. crispatus and Gardnerella/Prevotella/Fannyhessea at the same time in separate wells and examine their respective growth kinetics. Variations – Is the growth outcome different when a consortium of L. crispatus strains is used? If so, what is the optimal consortium? Can we create a L. crispatus consortium that is able to inhibit a consortium of BV-associated strains? Key metrics for all analyses – Growth kinetics

2) Is pre-incubation of the probiotic with the pathogen required for the induction of inhibitory activity? – Experiment: Grow duplicate cultures of L. crispatus to mid-log phase and then inoculate the second co-culture well in each pair with a BV-associated strain (‘induced’) or sterile media (‘uninduced’). Harvest the supernatant from each and use in a spot test for inhibitory activity. Key metrics – inhibition activity (plus some growth kinetics data)

3) What metabolomic changes occur when the probiotic candidates are co-cultured in the presence of BV- associated bacteria? Experiment: Inoculate either individual L. crispatus or strain consortia together with different BV-associated bacteria in the second co-culture well (along with ‘uninduced’ controls). Compare the expression profiles (rna-seq), proteomes (LC-MS) and metabolomes (LC-MS/MS) of L. crispatus under the two different conditions to identify possible substances responsible for the inhibitory activity.

Results from these experiments will help us to better understand interactions between vaginal bacteria and identify priority candidates for probiotic development.