subjects (#9, #24) with a continuous grade III VMF had a significantly higher (p,0.001) concentration of A. vaginae in comparison to G. vaginalis. In the other four group D women, for whom no or almost no grade III VMF episodes could be observed, A. vaginae was only sporadically present, with low average concentrations, varying between log4 and log5 cells/ml. Prevotella bivia. In Group N, 8 of 9 women were colonized by P. bivia around the menses and when the VMF was disturbed (grade I-like and II). Outside the menses, the concentrations of P. bivia, when present, varied between log4 and log6 cells/ml. During the menses, there was a 1–2 log increase, but it was only significant for one subject (p = 0.036). When the VMF remained disturbed after the menses (grade II VMF) or when the VMF shifted to grade II VMF, the concentrations increased to log9 cells/ml. In Group D, 5 of 8 women, i.e. the women with an overall grade Ib/II/III or continuous grade III VMF, were colonized by P. bivia (average 7 log7 cells/ml). For 4 of these women, the concentration increased by 1–2 logs during menses, but only significantly in one subject (p = 0.038). The woman (#18), with continuous grade II VMF during the study, was devoid of P. bivia, and the same was true for the subjects with grade Ib/II/III VMF, when they had episodes of grade II VMF. Comparison of the presence of the 5 species in relation to each other Within group N, L. crispatus was present on all tested days for all women (Figure 1), except for the two subjects with primarily grade Ib (#4 and #20). In group N, the daily presence of continuous high concentrations of L. crispatus (average log8 cells/ml) appeared to be protective against the prolonged presence of A. vaginae, P. bivia and G. vaginalis (sialidase positive and negative) (Figure 1). The two women with the most stable grade I VMF (almost no shifts between grades), i.e. subjects #14 and #15 (woman with no menses), had no A. vaginae and no sialidase positive G. vaginalis during the study and L. crispatus (#14) or L. crispatus and L. iners (#15) were the only species of the 5 tested that were constantly present. In group D (Figure 2), L. iners (average log9 cells/ml), A. vaginae (average log9 cells/ml), G. vaginalis (average log 9 cells/ml), sialidase positive G. vaginalis (average log8 cells/ml) and P. bivia (average log8 cells/ml) were present on all tested days of the two women (#9 and #24) with a continous grade III BV-associated VMF. In addition to these 4 species, the subjects (#21 and #25) shifting between grades Ib, II, III and IV VMF also had high concentrations (minimum log7 cells/ml) of L. crispatus, although L. crispatus was not present on all days. For subject #18, with grade II VMF throughout the study, all tested days were characterized by the presence of L. crispatus (average log8 cells/ml) and sialidase negative and sialidase positive G. vaginalis strains (average log9 cells/ml). Hydrogen-peroxide-production For 8 out of 9 women of group N, strong (score 2) to very strong (score 3) H2O2-production was observed. L. crispatus concentra- tions higher than log7 cells/ml characterized the VMF of all of these women . For the subjects, where a significant decrease in L. crispatus concentrations during the menses was observed, the H2O2-production was only negatively affected when the concen- trations dropped below log7 cells/ml, irrespective of the VMF grade. Exceptions were observed for subject #22 on two occasions, whereby the L. crispatus concentration remained high, but the H2O2-production dropped and for subject #20, with low concentrations of L. crispatus during the complete study period (average log6 cells/ml), but strong H2O2-production. However, Longitudinal qPCR Study of 5 Key Vaginal Species the latter subject was colonized by L. jensenii during the whole study, as determined by culture. In group D, 5 out of 8 women had none to very weak (score 0– 1) H2O2-production. These women were characterized by L. crispatus concentrations below log7 cells/ml. The three subjects with L. crispatus concentrations higher than log7 cells/ml had a strong H2O2-production. In general, when the concentrations of L. crispatus were below log7 and/or L. jensenii was absent, no or only weak H2O2- production was observed. The culture results confirm the importance of L. crispatus and L. jensenii. From a total of 178 culture moments, 108 moments presented with blue colonies on TMB+ agar, 73 of these 108 culture moments had a H2O2 score .1. L. crispatus and L. jensenii were present on respectively 36 and 40 of these 73 culture moments. In comparison, Streptococci where only present on 14 of these 78 culture moments. No L. crispatus was cultured from the 35 culture moments with a H2O2-score,1. File S2 contains detailed information about all species that colored blue on TMB+ agar, together with the culture moment and the corresponding H2O2 score and VMF grade. The influence of individual behavior on the presence of the 5 species In general, there was no clear influence of personal hygiene (bathing, shower, intimate hygiene, tampon use) on the presence of the 5 species. In group N, 7 incidences of sexual interaction (SI), of which 5 assessable, were documented for two subjects. For one subject, A. vaginae appeared (at ,log6 cells/ml) after SI with condom and the concentration of P. bivia increased (with 4 logs). For the other subject, after SI with condom, G. vaginalis appeared with concentrations between log5 and log6 cell/ml. In group D, 40 incidences of SI were documented for 4 subjects. However, because the VMF was already non-grade I or because SI occurred during the menses, only 7 instances of SI from one subject could be assessed. In this case, SI without condom resulted in the appearance of A. vaginae and G. vaginalis (sialidase negative) with concentrations between log5 and log6 cell/ml. The influence antibiotic therapy on the presence of the 5 species Subject #16 started the study with only L. iners, at log10–log11 cells/ml, and when the 5-day antibiotics cure was initiated, L. iners disappeared quickly. A. vaginae was present on one day during the antibiotics cure and it reappeared after the cure on two more occasions. P. bivia was not present before the antibiotic treatment, but it was the first species to appear 22 days after the treatment. L. iners reappeared only 30 days later at the end of the study. Subject #21 followed a 9-day antibiotics cure, whereby all 5 species remained present during the first 3 days. Thereafter, L. crispatus disappeared completely and only reappeared 3 weeks later. The concentrations of the other 4 species decreased more than 2 log, but these species recovered quickly. Discussion Overall findings In this study, L. crispatus was present in high concentrations when the VMF on Gram stain was grade Ia, Iab or II, and the concentrations clearly declined during the menses of the nine women, who were predominantly colonized by L. crispatus (7 from group N and 2 from group D). These results confirm the findings from previous studies [10,13,16]. The BV-associated bacteria, G. PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45281