The identification of body fluids through microbiological markers

Abstract

Microbial forensics has recently become the focus of many studies, since microbiota can provide useful information about a criminal case. The use of the human microbiome to identify perpetrators has been one of the major developments, due to the large number of bacteria present on and within the human body that are specific to the body habitat as well as to a particular individual. The identification of body fluids through bacterial DNA markers has thus been studied, since no appropriate confirmatory tests for certain fluids, such as saliva and vaginal fluid, have been developed to date. In this study, the suitability of Lactobacillus crispatus and L. iners as marker species for the identification of vaginal fluid was evaluated by means of a quantitative duplex real-time PCR targeting specific regions of the 16S rRNA gene. The standard curve method was tested on 109 vaginal fluid samples to determine whether the presence of the two species varies between individuals. Factors that might be influencing the occurrence of these lactobacilli have also been taken into consideration. The duplex reaction using hydrolysis probes for L. crispatus and L. iners was optimised, with the efficiencies recorded for the standard curves during validation being 99.96% and 99.71%, respectively, indicating that no non-specific products were being amplified. Nonetheless, L. crispatus was detected in 63.30% of the samples while L. iners was detected in 65.14%. Although L. crispatus and L. iners generally occurred together in the same individual, L. crispatus was typically found in higher quantities compared to L. iners. According to the logistic regression model, the presence of L. iners is more likely to increase if the person is not taking antibiotics, does not have an intrauterine device, is younger and hence pre-menopausal, and is on hormone replacement therapy. Antibiotic use also significantly reduced the likelihood of the presence of L. crispatus. Other trends that could be noted in the data were the enhanced presence of L. crispatus in women using oral contraceptives and the increased occurrence of both L. crispatus and L. iners in women in their third week of the cycle. The results suggest that using L. crispatus and L. iners as bacterial markers for vaginal fluid is not sufficient to confirm the presence of this fluid, since the occurrence of the two bacteria is influenced by numerous factors. Other bacterial or human marker genes should thus also be targeted such that this body fluid can be consistently detected in forensic casework.