Summary Few validated protocols are available for large-scale collection, storage, and analysis of microbiome samples from the vagina, skin, and mouth. To prepare for a large-scale study on the female microbiome by remote self-sampling, we investigated the impact of sample collection, storage, and host DNA depletion on microbiome profiling. Vaginal, skin, and saliva samples were analyzed using 16S rRNA gene amplicon and metagenomic shotgun sequencing, and qPCR. Of the two tested storage buffers, the eNAT buffer could keep the microbial composition stable during various conditions. All three tested host DNA-depletion approaches showed a bias against Gram-negative taxa. However, using the HostZERO Microbial DNA and QIAamp DNA Microbiome kits, samples still clustered according to body site and not by depletion approach. Therefore, our study showed the effectiveness of these methods in depleting host DNA. Yet, a suitable approach is recommended for each habitat studied based on microbial composition.

The vaginal microbiome is crucial for women’s health and reproduction, but its ecology and determinants in the general population are still unclear. This lack of a reference framework hampers much-needed innovations in diagnostics and therapeutics. Here, we remotely mapped the vaginal microbiome of 3,345 women in Western Europe via a citizen-science approach. More than 75% of the vaginal samples were dominated by Lactobacillus taxa, but not in discrete community state types. Compositional correlation network analysis validated with public data pointed at six main modules of interacting microbes: a Lactobacillus crispatus-, Lactobacillus iners-, Gardnerella-, Prevotella-, Anaerococcus-, and gut-derived module. In the first module, Limosilactobacillus taxa were functionally connected to L. crispatus and Lactobacillus jensenii. This module was positively associated with the luteal phase of the menstrual cycle and negatively with the number of vaginal complaints, while the Gardnerella-module was associated with discharge and increasing age. Contraceptives with oestrogen correlated with higher levels of the L. crispatus- and less of the Gardnerella-module, with the opposite found for a hormonal intrauterine device or having multiple partners. Mothers had lower relative abundance of the L. crispatus-module and more Bifidobacterium, Lactobacillus gasseri and Streptococcus. Other covariates such as BMI, menstrual pads and cups, smoking and dietary habits were also associated with the microbial constellation. These findings suggest that lifestyle interventions have potential to improve vaginal health when combined with dedicated therapies.

Microbial forensics represents a promising tool to strengthen traditional forensic investigative methods and fill related knowledge gaps. Large-scale microbiome studies indicate that microbial fingerprinting can assist forensics in areas such as trace evidence, source tracking, geolocation, and circumstances of death. Nevertheless, the majority of forensic microbiome studies focus on soil and internal organ samples, whereas the microbiome of skin, mouth, and especially vaginal samples that are routinely collected in sexual assault and femicide cases remain underexplored. This review discusses the current and emerging insights into vaginal, skin, and salivary microbiome-modulating factors during life (e.g., lifestyle and health status) and after death (e.g., environmental influences and post-mortem interval) based on next-generation sequencing. We specifically highlight the key aspects of female reproductive tract, skin, and mouth microbiome samples relevant in forensics. To fill the current knowledge gaps, future research should focus on the degree to which the post-mortem succession rate and profiles of vaginal, skin, and saliva microbiota are sensitive to abiotic and biotic factors, presence or absence of oxygen and other gases, and the nutrient richness of the environment. Application of this microbiome-related knowledge could provide valuable complementary data to strengthen forensic cases, for example, to shed light on the circumstances surrounding death with (post-mortem) microbial fingerprinting. Overall, this review synthesizes the present knowledge and aims to provide a framework to adequately comprehend the hurdles and potential application of vaginal, skin, and salivary post-mortem microbiomes in forensic investigations.

How to define and promote a healthy state of the vaginal microbiome is not well understood. Knowledge of which underlying factors shape the microbial community composition of the vagina and how to modulate them will contribute to vaginal disease prevention and improve fertility.

Riboflavin-producing lactic acid bacteria represent a promising and cost-effective strategy for food biofortification, but production levels are typically insufficient to support daily human requirements. In this study, we describe the novel human isolate Limosilactobacillus reuteri AMBV339 as a strong food biofortification candidate. This strain shows a high natural riboflavin (vitamin B2) overproduction of 18.36 μg/ml, biomass production up to 6 × 1010 colony-forming units/ml (in the typical range of model lactobacilli), and pH-lowering capacities to a pH as low as 4.03 in common plant-based (coconut, soy, and oat) and cow milk beverages when cultured up to 72 h at 37°C. These properties were especially pronounced in coconut beverage and butter milk fermentations, and were sustained in co-culture with the model starter Streptococcus thermophilus. Furthermore, L. reuteri AMBV339 grown in laboratory media or in a coconut beverage survived in gastric juice and in a simulated gastrointestinal dialysis model with colon phase (GIDM-colon system) inoculated with fecal material from a healthy volunteer. Passive transport of L. reuteri AMBV339-produced riboflavin occurred in the small intestinal and colon stage of the GIDM system, and active transport via intestinal epithelial Caco-2 monolayers was also demonstrated. L. reuteri AMBV339 did not cause fecal microbiome perturbations in the GIDM-colon system and inhibited enteric bacterial pathogens in vitro. Taken together, our data suggests that L. reuteri AMBV339 represents a promising candidate to provide riboflavin fortification of plant-based and dairy foods, and has a high application potential in the human gastrointestinal tract.