Development of a multiplex PCR assay and quantification of microbial markers by ddPCR for identification of saliva and vaginal fluid.

The identification of biological fluids at crime scenes contributes to crime scene reconstruction and provides investigative leads. Traditional methods for body fluid identification are limited in terms of sensitivity and are mostly presumptive. Emerging methods based on mRNA and DNA methylation require high quality template source. An exploitable characteristic of body fluids is their distinct microbial profiles allowing for the discrimination of body fluids based on microbiome content. Microbial DNA is highly abundant within the body, robust and stable and can persist in the environment long after human DNA has degraded. 16S rRNA sequencing is the gold standard for microbial analysis; however, NGS is costly, and requires intricate workflows and interpretation. Also, species level resolution is not always achievable. Based on the current challenges, the first objective of this study was to develop a multiplex conventional PCR assay to identify vaginal fluid and saliva by targeting species-specific 16S rRNA microbial markers. The second objective was to employ droplet digital PCR (ddPCR) as a novel approach to quantify bacterial species alone and in a mixture of body fluids. Lactobacillus crispatus and Streptococcus salivarius were selected because of high abundance within vaginal fluid and saliva respectively. While Fusobacterium nucleatum and Gardnerella vaginalis, though present in healthy humans, are also frequently found in oral and vaginal infections, respectively. The multiplex PCR assay detected L. crispatus and G. vaginalis in vaginal fluid while F. nucleatum and S. salivarius was detected in saliva. Multiplex PCR detected F. nucleatum, S. salivarius and L. crispatus in mixed body fluid samples while, G. vaginalis was undetected in mixtures containing vaginal fluid. For samples exposed at room temperature for 65 days, L. crispatus and G. vaginalis were detected in vaginal swabs while only S. salivarius was detected in saliva swabs. The limit of detection was 0.06 copies/µl for F. nucleatum (2.5 ×10-9 ng/µl) and S. salivarius (2.5 ×10-6 ng/µl). L. crispatus and G. vaginalis had detection limits of 0.16 copies/µl (2.5 ×10-4 ng/µl) and 0.48 copies/µl (2.5 ×10-7 ng/µl). All 4 bacterial species were detected in mixtures and aged samples by ddPCR. No significant differences were observed in quantity of bacterial markers in saliva and vaginal fluid. The present research reports for the first time the combination of the above four bacterial markers for the detection of saliva and vaginal fluid and highlights the sensitivity of ddPCR for bacterial quantification in pure and mixed body fluids.

Assessing time dependent changes in microbial composition of biological crime scene traces using microbial RNA markers.

Current body fluid identification methods do not reveal any information about the time since deposition (TsD) of biological traces, even though determining the age of traces could be crucial for the investigative process. To determine the utility of microbial RNA markers for TsD estimation, we examined RNA sequencing data from five forensically relevant body fluids (blood, menstrual blood, saliva, semen, and vaginal secretion) over seven time points, ranging from fresh to 1.5 years. One set of samples was stored indoors while another was exposed to outdoor conditions. In outdoor samples, we observed a consistent compositional shift, occurring after 4 weeks: this shift was characterized by an overall increase in non-human eukaryotic RNA and an overall decrease in prokaryotic RNA. In depth analyses showed a high fraction of tree, grass and fungal signatures, which are characteristic for the environment the samples were exposed to. When examining the prokaryotic fraction in more detail, three bacterial phyla were found to exhibit the largest changes in abundance, namely Actinobacteria, Proteobacteria and Firmicutes. More detailed analyses at the order level were done using a Lasso regression analysis to find a predictive subset of bacterial taxa. We found 26 bacterial orders to be indicative of sample age. Indoor samples did not reveal such a clear compositional change at the domain level: eukaryotic and prokaryotic abundance remained relatively stable across the assessed time period. Nonetheless, a Lasso regression analysis identified 32 bacterial orders exhibiting clear changes over time, enabling the prediction of TsD. For both indoor and outdoor samples, a larger number (around 60%) of the bacterial orders identified as indicative of TsD are part of the Actinobacteria, Proteobacteria and Firmicutes. In summary, we found that the observed changes across time are not primarily due to changes associated with body fluid specific bacteria but mostly due to accumulation of bacteria from the environment. Orders of these environmental bacteria could be evaluated for TsD prediction, considering the location and environment of the crime scene. However, further studies are needed to verify these findings, determine the applicability across samples, replicates, donors, and other variables, and also to further assess the effect of different seasons and locations on the samples.

The addition of Lactobacillus spp. negatively affects Mycoplasma bovis viability in bovine cervical mucus.

BACKGROUND: Mycoplasma bovis is an important pathogen for the cattle industry worldwide causing significant economic losses. Several transmission routes, including those related to reproduction, have been described. Indeed, the pathogen can colonize the female reproductive tract after artificial insemination (AI) with contaminated semen. Lactobacillus spp.-based probiotics have been used for vaginal dysbiosis treatment in women and cows although their role in controlling cervico-vaginal infections due to M. bovis is unknown. The objective of the present work is to assess the viability of M. bovis (PG45, NCTC 10131) in experimentally contaminated cervical mucus after the addition of Lactobacillus spp. at different concentrations as a competing agent and pH acidifier. RESULTS: The addition of probiotic at a concentration higher than 108 colony forming units (CFU/mL had a detrimental effect (P < 0.05) on mycoplasma viability in cervical mucus. This coincided with a significant LAB growth and an important decrease in pH from 8.4 to 5.6 (P < 0.05). However, after the addition of less concentrated probiotic, M. bovis survival was not affected and there was no significant LAB growth despite the drop of pH from 8.4 to 6.73 (P < 0.05). CONCLUSION: The addition of concentrations higher than 108 CFU/mL of Lactobacillus spp. negatively affects M. bovis viability in bovine cervical mucus under in vitro conditions. Although the effect observed on the pathogen viability seems to be related to the pH decrease after LAB proliferation in cervical mucus, further studies are necessary to elucidate if other factors are implicated. Nevertheless, the administration of Lactobacillus spp.-based probiotics might be used in the future to control M. bovis proliferation in the cervico-vaginal tract of cows.

Effect of indoor environmental exposure on seminal microbiota and its application in body fluid identification.

Semen is a common body fluid type in forensic sexual assault cases. It is of great significance to effectively identify semen for restoring the crime scene and determining the nature of the case. Nowadays, microbiome-based method shows as a promising tool for forensic body fluid identification. To explore the environmental impact on microbial community of semen and its traceability, 16S rDNA high-throughput sequencing was conducted to ten paired semen samples. Affected by exposure, the diversity of microbial community decreased generally as the genus Staphylococcus exhibited a relatively significant increase. However, the genus Staphylococcus, Corynebacterium, Corynebacterium_1 were observed in almost all 20 samples. Community barplot analysis and heatmap analysis showed composition of the predominant microbe in semen at the phyla and genus level maintained basically, so that it could distinguish from vaginal fluid and saliva regardless of environmental exposure. Based on these results, we believe the application of single microbial marker may limit in semen identification, but the method depending on microbial community might be useful for distinguishing semen even under indoor exposure.

Cervical-Vaginal Mucin in Fertility Assessment: CA125 as a Predictor of the Fertile Phase of the Normal Menstrual Cycle.

: To evaluate the cervical-vaginal mucin, CA125, as a measure of fertility and possible method for natural family planning (NFP). Cervical-vaginal fluid (CVF) swab samples have been previously used to measure CA125, 'Qvaginal CA125 levels', as a function of time of cycle relative to Day 0, the first day of positive urine LH (luteinizing hormone). Data from 15 women, 20 cycles were used with an algorithm to establish the Fertile Start Day (FSD) for the cycles. The FSD was determined as either the second consecutive day of ≥20% Qvaginal CA125 rise or the first day of ≥400% rise. The interval, (FSD to Day +3), was used as the theoretical window of fertility, and conception rates assuming abstinence during this predicted period of fertility were computed using published day-specific probabilities of conception (PoC). The mean FSD was Day -4.8 ± 0.5 (SE), 95% CI (-5.9, -3.7). The estimated pregnancy failure rate (PFR) with abstinence during [FSD, +3] was 10.7% ± 2.0% (SE), 95% CI (6.9%, 14.8%); with exclusion of one cycle with very low levels of Qvaginal CA125, the estimated PFR was 9.8% ± 1.9%, 95% CI (6.3%, 13.8%). Furthermore, the day-specific Qvaginal CA125 values were normalized to the respective peak Qvaginal CA125 for each cycle, and a mean normalized day-specific Qvaginal CA125 plot was generated. The first derivative of the mean normalized day-specific Qvaginal CA125 plot showed a significant increase between Day -4.5 and Day -3.5, which correlated with the mean FSD. A Qvaginal CA125-based method holds promise as a means to identify the start of the fertile window and may prove useful in NFP, especially when combined with available home hormonal fertility awareness kits.

A 7-year surveillance of the drug resistance in Klebsiella pneumoniae from a primary health care center.

BACKGROUND: The increased prevalence of Klebsiella pneumoniae infections and resistance rates are a current cause for concern. However, data for resistance rates in K. pneumoniae strains from primary hospitals and the resistance distribution among the different isolate sample sources are scarce. METHODS: All the K. pneumoniae strains were isolated from patients who visited a primary health care center located in Central Zhejiang Province from January 2011 to December 2017. The specimens included blood, sputum, cervical secretions and urine. The species were identified by the Vitek 2 Compact Bacterial Identification and Monitoring System or VITEK-MS and the extended spectrum ß-lactamase (ESBL) and drug resistance profiles were identified using the AST-GN13 Gram negative susceptibility card (VITEK-2). The genotype of strains from urine sources was analyzed by detecting TEM and SHV genes. Finally, the drug resistance rates among the isolates from different sample sources were analyzed using the Chi square test with SPSS software. RESULTS: A total of 5319 K. pneumoniae strains were isolated in this study. Among the 20 antimicrobial drugs studied, the resistance rates of K. pneumoniae strains varied from 1.4% (ertapenem) to 23.1% (nitrofurantoin). The antibiotic resistance rates varied significantly among the isolate samples sources for all, with the highest rates for all antibiotics except for nitrofurantoin found in urine samples. In addition, the ESBL-positive rate in urine samples was 27.1%, significantly higher than that of cervical secretions (20.2%), blood (16.5%) and sputum (15.2%). Compared to the ESBL-negative strains, higher resistance rates were detected in the ESBL-positive strains. The most common genotype of isolates from urine was SHV (28%, 23/82), following by TEM (14.6%, 12/82). CONCLUSION: The highest resistance rates of K. pneumoniae strains to most antibiotics found in urine samples are partly due to the ESBLs, indicating that a special attention should be paid in the treatment of urinary tract infection.

Identification of Campylobacter fetus subsp. venerealis virulence genes in cervical mucus from cows.

We used the polymerase chain reaction to identify virulence genes in cervico-vaginal mucus samples from cows positive for Campylobacter fetus subsp. venerealis. There was positivity for the pldA, racR, dnaJ, cdtA, and cdtB genes. No samples showed the cdtC, ciaB, cadF, wlaN, and virB11 genes.

Lactobacillus DNA usage in differentiation of normal vaginal fluids in premenopausal and postmenopausal females.

The presence of vaginal fluid as a bio-stain in the crime scene of sexual assaults provides pivotal evidence. The vaginal secretions are known to be rich in Lactobacillus; hence the current work aims to identify vaginal secretions via detection and quantification of Lactobacillus DNA in pre and postmenopausal females and to test its stability over storage time using Critical Threshold method applied by Polymerase chain reaction approach. Comparative study is done by Critical Threshold and Relative Expression methods aiming to evaluate the two methods. Results showed that (ΔCT) <9 powerfully indicates the presence of vaginal fluids. Values of ΔCT in all vaginal samples are stable and not affected by storage. Two novel cutoff values are obtained in order to differentiate between premenopausal and postmenopausal vaginal fluid samples which are (8.42) using the Critical Threshold method and (0.24) using the Relative Expression method. One novel cutoff value is obtained to differentiate between fresh and stored vaginal samples by the Relative Expression method which is (0.39). It is concluded that Lactobacillus DNA quantification via PCR is a good positive identifier for vaginal secretions which is remarkably stable over storage time.

Molecular detection of microbial colonization in cervical mucus of women with and without endometriosis.

PROBLEM: Intrauterine microbial colonization and its association with the pathogenesis of endometriosis via an innate immune cascade have been reported. As a potential source of microbial transmission, information on microbial colonization in cervical mucus is unknown. We investigated pattern of microbiota in the cervical mucus collected from women with and without endometriosis using next-generation sequencing (NGS) technology. METHOD OF STUDY: Cervical mucus samples were collected from women with (n = 30) and without (n = 39) endometriosis. The communities of microbiota in cervical mucus in the endometriosis group and the control group were examined by Gram staining and NGS targeting the V5-V6 region of 16S ribosomal RNA gene. Copy number of some target bacteria was detected by real-time PCR. RESULTS: We confirmed visual presence of bacteria in cervical mucus by Gram staining. NGS analysis showed that distribution of microbiota was similar in cervical mucus of women with and without endometriosis regardless of the phases of the menstrual cycle. In addition to predominant Lactobacilli spp., the populations of Corynebacterium, Enterobacteriaceae, Flavobacterium, Pseudomonas, and Streptococcus were increased in the endometriosis group. Of them, Enterobacteriaceae and Streptococcus were identified as the more significant candidates in the endometriosis group than in controls by real-time PCR (P < 0.05 for each). CONCLUSION: Our NGS analysis of cervical mucus indicated that among a variable microbiota, two candidates (Enterobacteriaceae and Streptococcus) were more frequently detected in women with endometriosis. Further investigation is needed to elucidate a mechanistic link of these bacteria in the pathophysiology of endometriosis.

Microbiome-based body fluid identification of samples exposed to indoor conditions.

In the forensic reconstruction of crime scene activities, the identification of biological traces and their bodily origin are valuable evidence that can be presented in court. While several presumptive and confirmatory tests are currently available, the limitations in specificity and sensitivity have instigated a search for alternative methods. Bacterial markers have been proposed as a novel approach for forensic body fluid/tissue identification. Bacteria are not only ubiquitous throughout the human body, but also, as shown by recent microbiome sequencing studies of the 16S rRNA gene, bacterial community structures are distinct across body sites. Traces and stains at crime scenes are, however, often exposed to the environment outside the human body for variable periods of time before laboratory processing. Thus, it is not clear whether exposed samples continue to harbor microbial signatures characteristic of their body site of origin. In this proof-of-concept study we collected samples from six different body sites: saliva, skin, peripheral blood, vaginal fluid, menstrual blood and semen. We exposed a subset of these samples to indoor conditions for 30 days while the remaining samples were processed directly after extraction. Our analyses of 16S rRNA gene sequence data for a total of 46 control and exposed samples show that both types of samples group by body site, although a few outliers are observed. Based on our results, vaginal and menstrual samples share their microbial signatures, and cannot be distinguished using bacterial markers. Overall, our findings indicate that bacterial markers are a promising avenue for forensic body fluid/tissue identification.

Specific microbes of saliva and vaginal fluid of Guangdong Han females based on 16S rDNA high-throughput sequencing.

Vaginal fluid and saliva are of great importance in forensic sciences. The identification of vaginal fluid or saliva is especially important in criminal cases. Microbes are considered as a promising marker for the identification of body fluids. In this study, 18 salivary fluids and 18 vaginal fluid samples were collected from 18 healthy women of the Han population in Guangdong province, China. The microbes of the above samples were analyzed by 16S rDNA high-throughput sequencing. The results showed that the microbes whose proportions are over 1% in saliva samples distributed across 12 genera and 57 operational taxonomic units (OTUs), and in vaginal fluid distributed across 4 genera and 9 OTUs. The microbes that dominated in saliva were quite different from those dominated in vaginal fluids. The linear discriminant analysis (LDA) effect size (LEfSe) algorithm was used to screen out the specific microbes of the studied samples, and the results showed that the specific microbes in saliva samples are Haemophilus parainfluenzae, Veillonella parvula, and Aggregatibacter segnis, while in vaginal fluid is Lactobacillus iners.

Cervico-vaginal mucus (CVM) - an accessible source of immunologically informative biomolecules.

Cervico-vaginal mucus (CVM), the product of epithelial cells lining the uterus, cervix and vagina, is secreted to facilitate uterine lubrication and microbial clearance. Predominantly composed of water and mucins, CVM also contains high levels of immuno-active proteins such as immunoglobulin A (IgA), lactoferrin and lysozyme which protect against infection by blocking adhesion and mediating microbial killing. The repertoire of cytokines, chemokines and antimicrobial peptides is predominantly generated by the secretions of endometrial epithelial cells into the uterine lumen and concentrated in the CVM. The quantity and relative proportions of these inflammatory biomarkers are affected by diverse factors including the estrus cycle and health status of the animal and therefore potentially provide important diagnostic and prognostic indicators. We propose that measuring molecular signatures in bovine CVM could be a useful approach to identifying and monitoring genital tract pathologies in beef and dairy cows.

Protein Content of Cervicovaginal Fluid Is Altered During Bacterial Vaginosis.

OBJECTIVES: The aim of the study was to compare, using a proteomic approach, cervicovaginal fluid (CVF) proteins of women with bacterial vaginosis (BV) with those presenting normal microbiota. MATERIALS AND METHODS: A total of 309 reproductive-aged women were cross-sectionally enrolled. Participants were tested for vaginal candidosis, Trichomonas vaginalis, Chlamydia trachomatis, and Neisseria gonorrhoeae and excluded if positive. Vaginal microbiota was classified microscopically according to Nugent criteria in normal, intermediate, and BV. Randomly selected CVF samples of 29 women with BV and an equal number with normal microbiota were subjected to proteomic analysis. Thus, a total of 58 CVF samples were evaluated using shotgun liquid chromatography-tandem mass spectrometry in a Q-Tof PREMIER API mass spectrometer (MicroMass/Waters) for peptide detection and relative quantification. RESULTS: Of the 309 women enrolled, 63 (20.4%) were excluded after testing positive for at least one of the tested co-infections or because of low-quality samples. Microscopic classification of vaginal microbiota on the remaining 246 samples revealed that 132 women (53.6%) had normal microbiota, 33 (13.4%) had intermediate microbiota, and 81 (33.0%) had BV. Proteomic analysis of CVF of 58 randomly selected women with normal microbiota (n = 29) or BV (n = 29) successfully identified 74 proteins. In addition, the comparison of abundance of those proteins between the groups showed that the following five (6.7%) were enriched in BV: neutrophil elastase, kaliocin-1, neutrophil defensin-1, Ig lambda-2 chain C regions, and protein S100-A7. All of which have a recognized role in host's immunity. CONCLUSIONS: Exclusive finding of BV affects immunity-related CVF components of reproductive-aged women.

Human Cervical Mucus Plugs Exhibit Insufficiencies in Antimicrobial Activity Towards Group B Streptococcus.

Preterm birth is a leading cause of neonatal mortality and lacks an effective therapy. Ascending microbial infections from the lower genital tract lead to infection of the placenta, amniotic fluid, and fetus causing preterm birth or stillbirth. Directly in the path of an ascending infection is the cervical mucus plug (CMP), a dense mucoid structure in the cervical canal with potential antimicrobial properties. In this study, we aimed to define the components of CMP responsible for antimicrobial activity against a common lower genital tract organism associated with preterm birth and stillbirths, namely, group B streptococcus (GBS). Using a quantitative proteomic approach, we identified antimicrobial factors in CMPs that were collected from healthy human pregnancies. However, we noted that the concentration of antimicrobial peptides present in the human CMPs were insufficient to directly kill GBS, and antimicrobial activity, when observed, was due to antibiotics retained in the CMPs. Despite this insufficiency, CMP proteins were able to activate leukocytes in whole blood resulting in increased rates of bacterial killing, suggesting a role for the CMP in enhancing complement-mediated killing or leukocyte activation. This study provides new insight into how the human CMP may limit ascending bacterial infection.

Assessment of cow and farm level risk factors associated with Ureaplasma diversum in pasture-based dairy systems - A field study.

Potential risk factors for Ureaplasma diversum in the vaginal mucus of 1,238 dairy cows were included in a multivariate logistic regression model, based on the cow level (i.e., granular vulvovaginitis [+GVV], yearly milk production [4500 kg or more], pregnancy, predominance of Bos taurus [+Bos Taurus], score of corporal condition [at least 2.5], concomitant positivity for Escherichia coli [+E.coli]), and farm level i.e., milking room hygiene (-Milking room), dunghill location, and replacement female). Ureaplasma diversum was present in 41.1% of the samples. Independent risk factors for U. diversum were +GVV (odds ratio [OR], 1.31); +Mycoplasma spp (OR, 5.67); yearly milk production (4500 kg or more) (OR, 1.99); +Bos taurus (OR, 1.68); +E. coli (OR, 4.96); -milking room (OR, 2.31); and replacement females (OR, 1.89). Ureaplasma diversum vaginal colonization was strongly associated with Mycoplasma spp., E. coli, and number of pregnant cows.

Assessment of cow and farm level risk factors associated with Ureaplasma diversum in pasture-based dairy systems - A field study

ABSTRACT Potential risk factors for Ureaplasma diversum in the vaginal mucus of 1,238 dairy cows were included in a multivariate logistic regression model, based on the cow level (i.e., granular vulvovaginitis [+GVV], yearly milk production [4500 kg or more], pregnancy, predominance of Bos taurus [+Bos Taurus], score of corporal condition [at least 2.5], concomitant positivity for Escherichia coli [+E.coli]), and farm level i.e., milking room hygiene (-Milking room), dunghill location, and replacement female). Ureaplasma diversum was present in 41.1% of the samples. Independent risk factors for U. diversum were +GVV (odds ratio [OR], 1.31); +Mycoplasma spp (OR, 5.67); yearly milk production (4500 kg or more) (OR, 1.99); +Bos taurus (OR, 1.68); +E. coli (OR, 4.96); -milking room (OR, 2.31); and replacement females (OR, 1.89). Ureaplasma diversum vaginal colonization was strongly associated with Mycoplasma spp., E. coli, and number of pregnant cows.

Beneficial effects of a Coriolus versicolor-based vaginal gel on cervical epithelization, vaginal microbiota and vaginal health: a pilot study in asymptomatic women.

BACKGROUND: To assess the effect of a 12-day treatment using a vaginal gel based on niosomes containing hyaluronic acid, ß-glucan, alpha-glucan oligosaccharide, Coriolus versicolor, Asian centella, Azadirachta indica and Aloe vera on vaginal microbiota, cervical epithelization and vaginal health. METHODS: Open-label, prospective pilot study conducted in asymptomatic women in daily practice. Cervical epithelization was evaluated by colposcopy using an ectopy epithelization score (from 5: no ectopy to 1: severe ectopy and bleeding), vaginal microbiota using the VaginaStatus-Diagnostic test (Instiüt für Mikroökologie, Herborn, Germany) and further rated by the investigator using a 5-point Liker scale (from 5: normal to 1: very severe deterioration in which all evaluated species were altered), and vaginal health using the Vaginal Health Index. RESULTS: In 21 women, a positive effect to improve epithelization of the cervical mucosa, with a mean score of 4.42 at the final visit as compared to 3.09 at baseline (P < 0.0001) (43% improvement). In 10 women, there was a trend of improving of vaginal microbiota status, with a mean score of 4.0 at the final visit vs. 3.3 at baseline (P = NS) (21.2% improvement). In 11 women, the Vaginal Health Index increased from 19.0 at baseline to 22.3 at the final visit (P = 0.007). The concentration of Lactobacillus spp. increased 54.5% of women and pH decreased from 4.32 to 4.09. CONCLUSIONS: These encouraging preliminary results provide the basis for designing a randomized controlled study, and for potential use in human papilloma virus infection. TRIAL REGISTRATION: ISRCTN77955077 . Registration date: February 15, 2017. Retrospectively registered.

Informativeness of NGS Analysis for Vaginal Fluid Identification.

The identification of vaginal fluids in forensic examinations plays an important role in crime scene reconstruction. Molecular detection of vaginal bacterial communities can lead to the correct discrimination of body fluids. These kinds of studies can be performed through multiplex real-time PCR using primers for a specific selection of bacteria. The availability of next-generation sequencing (NGS) protocols provided for the extension of the analysis to evaluate the prokaryotes present in specimens. In this study, DNA was extracted from 18 samples (vaginal, oral, fecal, yoghurt) and analyzed by real-time PCR and NGS. The comparison between the two approaches has demonstrated that the information developed through NGS can augment the more conventional real-time PCR detection of a few key bacterial species to provide a more probative result and the correct identification of vaginal fluid from samples that are more forensically challenged.

Distinct Ecological Niche of Anal, Oral, and Cervical Mucosal Microbiomes in Adolescent Women.

Human body sites represent ecological niches for microorganisms, each providing variations in microbial exposure, nutrient availability, microbial competition, and host immunological responses. In this study, we investigated the oral, anal, and cervical microbiomes from the same 20 sexually active adolescent females, using culture-independent, next-generation sequencing. DNA from each sample was amplified for the bacterial 16S rRNA gene and sequenced on an Illumina platform using paired-end reads. Across the three anatomical niches, we found significant differences in bacterial community composition and diversity. Overall anal samples were dominated with Prevotella and Bacteriodes, oral samples with Streptococcus and Prevotella, and cervical samples with Lactobacillus. The microbiomes of a few cervical samples clustered with anal samples in weighted principal coordinate analyses, due in part to a higher proportion of Prevotella in those samples. Additionally, cervical samples had the lowest alpha diversity. Our results demonstrate the occurrence of distinct microbial communities across body sites within the same individual.

Identification of vaginal fluid, saliva, and feces using microbial signatures in a Han Chinese population.

In recent years, forensic scientists have focused on the discrimination of body fluids using microbial signatures. In this study, we performed PCR-based detection of microbial signatures of vaginal fluid, saliva, and feces in a Han Chinese population. We investigated the 16S rRNA genes of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, and Atopobium vaginae in vaginal fluid, the 16S rRNA and the glucosyltransferase enzyme genes of Streptococcus salivarius and Streptococcus mutans in saliva, and the 16S rRNA genes of Enterococcus species, the RNA polymerase ß-subunit gene of Bacteroides uniformis and Bacteroides vulgatus, and the α-1-6 mannanase gene of Bacteroides thetaiotaomicron in feces. As a result, the detection proportions of L. crispatus, L. gasseri, L. jensenii, L. iners, and A. vaginae were 15/16, 5/16, 8/16, 14/16, and 3/16 in 16 vaginal fluid donors, respectively. L. crispatus and L. jensenii were specifically detected in vaginal fluid; L. gasseri, L. iners, and A. vaginae were also detected in non-vaginal fluid. S. salivarius and S. mutans were not specifically detected in saliva. The detection proportions of Enterococcus species, B. uniformis, B. vulgatus, and B. thetaiotaomicron in 16 feces samples were 16/16, 12/16, 15/16, and 11/16, respectively. B. uniformis and B. thetaiotaomicron were specifically detected in feces. In addition, DNA samples prepared for the identification of body fluid can also be used for individual identification by short tandem repeat typing. The mean detection sensitivities of L. crispatus and L. jensenii were 0.362 and 0.249 pg/uL, respectively. In conclusion, L. crispatus, L. jensenii, B. uniformis, and B. thetaiotaomicron can be used as effective markers for forensic identification of vaginal fluid and feces.