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Background: Signature matching of nucleotide sequences in the V1 and V3 regions 16S rRNA genes using pyrosequencing technology is a powerful tool for typing vaginal Lactobacilli to the species level and has been used for investigating the vaginal microbial niche., Methods: This study has characterized the normal cultivable vaginal Lactobacillus flora at varying estradiol levels in plasma; the study comprised 17 patients undergoing ovarian stimulation for In Vitro Fertilization (IVF) treatment. The vaginal status of each participant was initially assessed as normal according to Amsel and Nugent criteria., Results: L. crispatus, L. gasseri and/or L. jensenii were present in 10 of the patients throughout the study period, and little variation among these three species was encountered in individual patients. The flora of three women was dominated by L. delbrüeckii, L. rhamnosus or L. vaginalis. One woman exhibited a dominance of L. iners. The flora of the remaining three women were initially dominated by L. rhamnosus or L. reuteri, but as their estrogen levels rose, their flora composition altered, to become dominated by one of the three species most common in a normal, healthy vagina., Conclusion: Signature matching of nucleotide sequences in the V1 and V3 regions of 16S rRNA genes is a discriminative tool for the study of vaginal Lactobacilli and can be used to track the Lactobacillus flora under a variety of physiological conditions.

Background: The diagnosis of bacterial vaginosis (BV) is often made according to Nugent's classification, a scoring system based on bacterial counting of Gram stained slides of vaginal secretion. However as the image area of the microscope field will influence the number of morphotypes seen there is a need to standardise the area., Methods: A graph intended for recalculation of number of bacterial morphotypes seen by the observer using 1000 x magnification from various microscope set-ups was constructed and applied to data sets typical for scoring BV. The graph was used in recalculation of Nugent scores, which were also compared with the Ison/Hay scores to evaluate the consequences for the diagnosis of BV., Results: The observed image area differed by 300% among the investigated microscope set-ups. In two different data sets, one treatment study and one screening study, a considerable change in the number of women classified as intermediate was seen when the graph was used to standardise the image area. The recalculated numbers were also compared to the Ison/Hay classification. Weighted kappa indexes between the different methods were 0.84, 0.88, and 0.90, indicating that the methods are comparable., Conclusion: Because of the considerable differences among image areas covered by different microscope set-ups used in Nugent and Ison/Hay scoring, there is a need to standardise the area in order to reach comparable scores reflecting the diagnosis of BV in different laboratories. The differences in the intermediate group will have a considerable effect on the results from both treatment and prevalence studies, even though the kappa indexes indicate very good agreement between the methods used.

The present study aimed to characterize lactobacilli in vaginal fluid from 23 adult healthy women by using high-throughput DNA sequencing for identification of a large number of randomly selected colonies appearing on Rogosa and blood agar. The typing method was based on broad-range PCR of 16S rRNA gene variable regions V1 and V3, pyrosequencing, and classification of the fragments by alignment with NCBI-catalogued sequences and type strain sequences. Four major groups of sequences were found among the 402 isolates clearly corresponding to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii when compared to the sequences obtained for type strains. Our results indicate that pyrosequencing of 16S rRNA gene fragments as used here is a fast and reliable method well suited for identification to the species level, even within the Lactobacillus acidophilus complex.

Species of the Lactobacillus acidophilus complex are generally considered to constitute most of the vaginal Lactobacillus flora, but the flora varies between studies. However, this may be due to difficulties in identifying the closely related species within the L. acidophilus complex by using traditional methods and to variations in the vaginal status of the participants. Two hundred two isolates from the vaginal fluids of 23 Swedish women without bacterial vaginosis, as defined by the criteria of Nugent et al. (R. P. Nugent, M. A. Krohn, and S. L. Hillier, J. Clin. Microbiol. 29:297-301, 1991), were typed by randomly amplified polymorphic DNA (RAPD) analysis and identified to the species level by temporal temperature gradient gel electrophoresis, multiplex PCR, and 16S ribosomal DNA sequencing. The vaginal flora of most participants was dominated by a single RAPD type, but five of them harbored two RAPD types representing two different species or strains. The most frequently occurring species were Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii. L. iners has not previously been reported as one of the predominant Lactobacillus species in the vagina.