inst a local database of 3 unique, full-length G. vaginalis 16S rRNA gene sequences acquired from the Ribosomal Database Project. We used a Shannon entropy-based approach to identify nucleotide positions that exhibit a high level of variation, and concatenated these nucleotides to define a set of 46 “oligotypes”. We examined patterns in the distribution and relative abundance of these oligotypes within individual couples, as well as across genders, anatomical sampling sites, and GS-defined BV and normal microbiota.
Materials and Methods
All patients enrolled in this study signed written informed consent to their participation. The study protocol and consent form was approved by the LSU Health Sciences Center Institutional Review Board.
Sample collection and clinical measurements
53 monogamous heterosexual couples were included in this study. The couples were recruited at the New Orleans STD clinic. From these 53 couples, we obtained 157 DNA samples (2 males did not provide urethral swabs). All subjects were at least 18 years old with no history of antibiotic use in the past 28 days, and couples presented together for evaluation. A vaginal swab was collected from each woman for DNA extraction and pyrosequencing analysis of bacterial composition. A separate vaginal swab sample was collected and characterized by GS NS . The samples were designated “normal” (NS = 0–3), “intermediate” (NS = 4–6) or “BV” (NS = 7–10). Two urethral swabs and two penile skin swabs were collected from males. For penile skin samples, two sterile Copan flocked swabs were used. One was rolled with firm pressure around the circumference of the coronal sulcus and over the surface of the glans penis. The second one was rolled with firm pressure all over the penile shaft. Urethral swabs were collected by inserting a sterile swab into the urethral meatus and rotating back and forth for 2–3 seconds. The first urethral swab was rolled on a slide and stained with a modified methylene blue stain to evaluate for the presence of urethritis. The penile skin and second urethral swabs were immediately placed in individual sterile tubes containing 3 ml of DNA preservative (GeneLockTM, Sierra Molecular Corp., Sonora, CA).
Extraction of DNA from swab samples was performed using commercial kits according to the manufacturer's instructions. An initial bacterial cell lysis step using lysozyme (20 mg/ml at 37°C for 1 hour) was included (QIAamp DNA micro kit for male, QIAamp DNA mini kit for female samples, Qiagen Inc., Valencia, CA). DNA obtained from the coronal sulcus and penile shaft swabs was combined for the analyses of bacterial composition of penile skin. Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) was performed by the Research and Testing Laboratory (Lubbock, TX) using broad-range PCR-amplification of the approximately 570bp long V4 -V6 region of the 16S rRNA gene with primers 530F: GTGCCAGCMGCNGCGG and 1100R: GGGTTNCGNTCGTTG. Due to the difficulty extracting DNA from penile skin and urethra samples, amount of DNA per PCR reaction ranged from 1ng to 25ng (25ng per vaginal sample, 10ng per urethra sample, 1ng to 5ng per penile skin sample).
Pyrosequencing analysis and extracting G. vaginalis sequences
Pyrosequencing analysis of all samples generated a total of 1,106,703 reads from 157 DNA samples. Of the total reads, 14.48% were discarded during the quality control step; 112,537 of these were short sequences (<200bp), 44,925 had one or more ambiguous bases, 1,022 had a mean quality score below Q25, and 1,838 had a single homopolymer region longer than 6 nucleotides. The average length of resulting 946,381 sequences that passed quality control was 481 nucleotides, with a standard deviation of 71, and the average number of sequences per sample was 6,257 with a standard deviation of 3,518. In order to identify and segregate the G. vaginalis reads from the rest of the sequences in the pyrosequencing library, we created a local database using three unique full-length G. vaginalis 16S rRNA gene sequences, acquired from the Ribosomal Database Project (the 