Changes in the microbial community after vaginal fluid exposure in different simulated forensic situations.

If vaginal fluid is found on clothing or on the body of the suspect, it may indicate the occurrence of sexual assault. Therefore, it is important to collect the victim's vaginal fluid at different sites from the suspect. Previous studies have revealed that fresh vaginal fluids can be identified based on 16S rRNA gene sequencing data. However, the influence of environmental factors on the stability of microbial markers must be investigated before being used in forensic practice. We collected vaginal fluid from nine unrelated individuals and placed each individual of vaginal swab on five different substrates. A total of 54 vaginal swabs were analyzed using 16S rRNA on the V3-V4 regions. Then, we constructed a random forest model including the samples of all vaginal fluids in this study and the other four types of body fluids in our previous studies. The alpha diversity of vaginal samples increased after exposure to the substrate environment for 30 days. The dominant vaginal bacteria were Lactobacillus and Gardnerella, which remained relatively stable after exposure, with Lactobacillus being the most abundant in all substrates, while Gardnerella was more abundant in other substrates than in the polyester fiber substrate. Except for bed sheets, Bifidobacterium significantly declined when placed on other substrates. Rhodococcus and Delftia from the substrate environment migrated to the vaginal samples. Rhodococcus was abundant in polyester fibers, and Delftia was abundant in wool substrates, while those environmental bacteria were all in low abundance in bed sheets. Overall, the bed sheet substrates showed a good retention capacity for the dominant flora and could reduce the number of taxa migrated by the environment compared with the other substrates. Both fresh and exposed vaginal samples of the same individuals could mostly be clustered and clearly distinguished from different individuals, showing the potential of individual identification, and the confusion matrix value of body fluid identification for vaginal samples was 1. In summary, vaginal samples placed on the surface of different substrates retained their stability and demonstrated good application potential for individual and body fluid identification.