Quantitative determination by real-time PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners

Background Most studies of the vaginal microflora have been based on culture or on qualitative molecular techniques. Here we applied existing real-time PCR formats for Lactobacillus crispatus, L. gasseri and Gardnerella vaginalis and developed new formats for Atopobium vaginae, L. iners and L. jensenii to obtain a quantitative non culture-based determination of these species in 71 vaginal samples from 32 pregnant and 28 non-pregnant women aged between 18 and 45 years. Results The 71 vaginal microflora samples of these women were categorized, using the Ison and Hay criteria, as refined by Verhelst et al. (2005), as follows: grade Ia: 8 samples, grade Iab: 10, grade Ib: 13, grade I-like: 10, grade II: 11, grade III: 12 and grade IV: 7. L. crispatus was found in all but 5 samples and was the most frequent Lactobacillus species detected. A significantly lower concentration of L. crispatus was found in grades II (p < 0.0001) and III (p = 0.002) compared to grade I. L. jensenii was found in all grades but showed higher concentration in grade Iab than in grade Ia (p = 0.024). A. vaginae and G. vaginalis were present in high concentrations in grade III, with log10 median concentrations (log10 MC), respectively of 9.0 and 9.2 cells/ml. Twenty (38.5%) of the 52 G. vaginalis positive samples were also positive for A. vaginae. In grade II we found almost no L. iners (log10 MC: 0/ml) but a high concentration of L. gasseri (log10 MC: 8.7/ml). By contrast, in grade III we found a high concentration of L. iners (log10 MC: 8.3/ml) and a low concentration of L. gasseri (log10 MC: 0/ml). These results show a negative association between L. gasseri and L. iners (r = -0.397, p = 0.001) and between L. gasseri and A. vaginae (r = -0.408, p < 0.0001). Conclusion In our study we found a clear negative association between L. iners and L. gasseri and between A. vaginae and L. gasseri. Our results do not provide support for the generally held proposition that grade II is an intermediate stage between grades I and III, because L. gasseri, abundant in grade II is not predominant in grade III, whereas L. iners, abundant in grade III is present only in low numbers in grade II samples.


Background
Bacterial vaginosis (BV) is considered to be the most frequent vaginal infectious disorder in women of childbearing age. Prevalences of 4.9% to 36% have been reported from European and American studies [1]. The condition is symptomatic in half of the women and also represents a psychological burden. More importantly, this disturbed vaginal microflora can cause serious sequelae such as nongonococcal, non-chlamydial PID (Pelvic Inflammatory Disease) [2,3], postpartum endometritis [4] and preterm birth [5][6][7]. Indeed, 40% of the cases of spontaneous preterm labor and preterm birth are thought to be associated with BV [8]. BV has also been associated with increased susceptibility to HIV and to genital tract infection with Chlamydia trachomatis and Neisseria gonorrhoeae [9][10][11].
BV is characterized by an overgrowth of many different, mostly anaerobic, bacteria. Gardnerella vaginalis has been considered to be the most characteristic microorganism in BV, but its pathogenic role was unclear, until recently, when the establishment of bacterial biofilm as a potential cause of recurrent BV [12], with a predominant presence of G. vaginalis, has clearly established a pathogenic role for this bacterium. Also recently, Atopobium vaginae has been strongly associated with BV, independently by different groups [13][14][15] and with biofilm in BV [12]. The suggestion that its association with BV is stronger than that of G. vaginalis, basically because G. vaginalis is more frequently detected in a normal vaginal microflora than A. vaginae [14] was confirmed by more recent studies [16,17]. The insight that biofilm is formed in BV [12] has strong explanatory power with regard to the high recurrence rate of BV, despite initial relief after antibiotic treatment [18] and is in perfect correlation with the presence of the characteristic cells or 'clue cells', which are vaginal epithelial cells covered with layers of bacteria.
Four species of lactobacilli are now considered to be predominantly linked to the vaginal microflora: Lactobacillus crispatus, L. jensenii, L. gasseri and L. iners, with the latter only recently being recognized as it was long overlooked because it does not grow on De Man Rogosa Sharpe agar, the medium typically used to culture lactobacilli [19,20].
In 1983 Spiegel et al. [21] devised a method for the diagnosis of bacterial vaginosis by direct Gram stain. Nugent et al. [22] proposed a modification of these criteria. By counting the different cell types (Lactobacillus spp., Gardnerella vaginalis/Bacteroides spp., Mobiluncus spp.) a score between zero and ten is obtained, whereby a score of 7 or higher corresponds to bacterial vaginosis and a score between 3 and 7 is considered intermediate between undisturbed vaginal microflora and bacterial vaginosis. Ison and Hay [23] suggested a simplification of this timeconsuming approach, through an estimation of the ratios of the observed cellular types rather than the determination of the exact number of the bacteria and distinguished five grades (0 (no bacteria), I (normal), II (intermediate), III (bacterial vaginosis) and IV (streptococci)). Verhelst et al. [24] recently suggested, on the basis of Gram stain, terminal restriction fragment length polymorphism analysis of the 16S rRNA gene (T-RFLP) and molecular identification (tDNA-PCR) of cultured isolates, a new classification of the undisturbed vaginal microflora (grade I) whereby grade I microflora could be split up into four categories, designated grade Ia, Ib, Iab and I-like. Grade Ia was shown to contain predominantly L. crispatus, grade Ib predominantly L. gasseri and L. iners, grade Iab a mixture of these three species and grade I-like Bifidobacterium spp. rather than Lactobacillus spp. Grade I-like microflora, of which the Gram stain at first glance indicates the presence of lactobacilli, was considered as not representative for normal vaginal micoflora, but as probably an unrecognized type of disturbed vaginal microflora [25] that had previously not been distinguished from a healthy vaginal microflora.
In this study, we developed real-time PCR primers for L. iners, L. jensenii and A. vaginae and used these, together with described real-time PCR formats for L. crispatus [26], L. gasseri [26] and G. vaginalis [27], in an attempt to quantify some of the important bacterial species in the normal and disturbed vaginal microflora.

Grading of vaginal microflora
In this study, Gram-stained smears of 71 vaginal swabs from 60 women, 32 of whom were pregnant, were examined microscopically. For 11 pregnant women the Gram stain based grading of their vaginal microflora differed between two trimesters and therefore swabs from both trimesters were included. Eight women had a vaginal microflora categorized by Gram stain as grade Ia, 10 as grade Iab, 13 as grade Ib, 10 as grade I-like, 11 as grade II, 12 as grade III and 7 as grade IV, according to criteria described previously [24].

Primer development and real-time PCR format
Primers for real-time PCR for Atopobium vaginae, Lactobacillus iners and L. jensenii were developed during this study (Tables 1, 2, 3). Primer positions relative to the 16S rRNA gene of Escherichia coli are presented in Figure 1. Table 4 lists all primers used and the real-time PCR formats. No cross-reactivity was detected with DNA from any of the tested species, including closely-related Lactobacillus and Atopobium species. In addition we found no cross reactivity for Bifidobacterium bifidum, B. breve and B. infantis with the G. vaginalis primers developed by Zariffard et al. [27].
Using Qiagen extracted DNA from pure cultures; the detection limit for A. vaginae is 1.3 pg/ml, for G. vaginalis 43.2 pg/ml, for L. crispatus 4.7 pg/ml, for L. gasseri 10.7 pg/ml, for L. iners 6.4 pg/ml and for L. jensenii 15.0 pg/ ml. Table 5 presents the overall presence of the six species studied in the different grades of vaginal microflora. L. crispatus was found in all but 5 samples (93%) and was the most frequent Lactobacillus species detected. L. jensenii was found in 33 samples (46%) but especially in grade Iab where it was present in 8 of 10 samples (80%).

Qualitative results
Forty-eight samples (68%) were positive for G. vaginalis and 20/48 (42%) of these were also positive for A. vaginae. Only three samples were positive for A. vaginae but negative for G. vaginalis. All grade III samples were positive for G. vaginalis and 10/13 (83%) were also positive for A. vaginae.

Quantitative results
The median log 10 cells/ml were expressed as per 1 ml elution buffer. We found a significantly higher level of L. crispatus in grade I (log 10 Median Concentration (MC) = 8.8 cells/ml) compared to the other grades, i.e. II: 4.2, p < 0.0001, III:
L. jensenii was found in all grades but showed higher concentration in grade Iab (log 10 MC = 7.0 cells/ml) than in grade Ia (log 10 MC = 0 cells/ml, p = 0.024) The level of L. gasseri in grade I (log 10 MC = 6.6 cells/ml) was significantly lower than in grade II (log 10 MC = 8.7 cells/ml, p < 0.01) and significantly higher than in grade III (log 10 MC = 0 cells/ml, p < 0.0001). The quantity of L. gasseri in grade II was significantly higher than in grade III (p < 0.0001) and than in grade IV (log 10 MC = 0 cells/ml, p < 0.05).
The level for L. iners did not differ either between grades I and III or between grades I and I-like. L. iners was present in grade II (log 10 MC = 0 cells/ml) in significantly lower amounts than in grade III (log 10 MC = 8.3 cells/ml, p < 0.0001), in grade I (log 10 MC = 6.5 cells/ml, p < 0.0001), in grade IV (log 10 MC = 4.3 cells/ml, p < 0.05) and in grade I-like (log 10 MC = 4.3 cells/ml, p < 0.05).
The concentrations of A. vaginae and G. vaginalis were significantly higher in grade III (respectively log 10 MC = 9.0
Gram staining of L. iners and L. gasseri Figure 3 represents the Gram stains of 6 strains of L. gasseri and 6 strains of L. iners. Gram stains of L. iners and L. gasseri showed that many of these strains displayed pleiomorphic cell morphology, i.e. different cell types could be observed within one microscopic field. Also, the cell morphologies of strains differed within the same species. Most strikingly, all L. iners strains had a relatively Gram-negative stain appearance and were mostly very short rods. By contrast, the L. gasseri were clearly Gram-positive in all cases. Some strains of L. gasseri formed streptobacillar chains, consisting of long to extra-long rods.

MIC of L. gasseri and L. iners for clindamycin
The clindamycin MIC value for ten strains of L. iners and L. gasseri was determined using the agar dilution method. Eight of the ten L. iners had a MIC value of 0.125 μg/ml, one strain had a MIC value of 2 μg/ml and one strain of >8 μg/ml. For L. gasseri one strain had a MIC value of 0.5 μg/ml, two strains had MIC values of 2 μg/ml, six strains had MIC values of 4 μg/ml and for one strain the MIC was more than 8 μg/ml. In summary, 80% of L. iners isolates were inhibited by 0.125 μg of clindamycin/ml, with MIC 50 = 0.125 μg/ml, whereas 90% of the L. gasseri isolates was resistant to 1 μg of clindamycin/ml, with MIC 50 = 4 μg/ml.

Mutual inhibition of L. gasseri and L. iners
No inhibition zones were observed with the techniques we used.

Discussion
In this study we used real-time PCR to obtain a quantitative estimation of the presence of the four predominant Lactobacillus species known to occupy the vaginal econiche and of Atopobium vaginae and Gardnerella vaginalis.  Localisation of the primers used in this study on the 16S rRNA gene Figure 1 Localisation of the primers used in this study on the 16S rRNA gene. Compared to microscopy and culture, molecular methods such as PCR may provide less observer-dependent and culture medium dependent information and a more Overview of the results obtained with real-time PCR for L. crispatus, L. jensenii, L. gasseri, L. iners, G. vaginalis and A. vaginae direct and detailed view of the quantity of certain species in a sample. E.g., A. vaginae was only recently recognized as strongly linked to disturbed vaginal microflora because of its fastidious growth requirements in vitro. A possible additional advantage of PCR, compared to culture, is that it permits detection of dead or metabolically inactive bacteria, and as such it can be applied for the detection of bacteria in a dormant state, as is the case in biofilms, which occur also in BV as has been shown by means of FISH [12].
Besides culture-associated bias, difficulties with regard to the standardization of vaginal sampling make quantification of the vaginal microflora cumbersome and make bacterial counts reported in different publications difficult to compare. Some groups have attempted to standardize sampling by cervicovaginal lavage (CVL), whereby the volume of saline that is recovered from the vagina can be measured and used for calibration of the bacterial load/ ml. Drawbacks are that CVL samples also from the cervix and does not represent a pure vaginal sample. Also it cannot be excluded that biofilm, that may be present in cases of BV, is recalcitrant with regard to dissolving in the saline that is used and is not sampled in the same manner as loosely associated microflora. In the condition of BV there may be more vaginal cell debris so it is feasible that there is more non-bacterial material present in the sample. In conclusion, vaginal sampling remains difficult to standardize. In this study we used unweighted vaginal swabs taken by the same gynecologist so there is no standardization for the volume of the sample, but standardization of the way samples are taken.

Microscopic images (100 ×) of Gram stains of L. iners and L. gasseri
Besides sampling bias and culture bias also real-time PCR may be biased, because the conversion of the PCR positivity threshold value to a concentration of bacteria, depends on the manner the standard curve is calibrated. The standard curve, which is prepared by extracting DNA from a dense bacterial suspension and by serial tenfold dilution of this DNA extract, can be compared to i) the bacterial colony count, as determined by plating out tenfold serial dilutions of the initial suspension, or to ii) the number of bacterial genomes in the initial suspension, determined on the basis of measurement of the DNA-concentration present in the extract from the initial suspension. We found differences between calculation of number of genomes by measurement of DNA-concentration and determination of number of cells by dilution culture from less to one log 10 unit (L. jensenii) to more than 3 log 10 units (L. crispatus) (data not presented). A possible explanation may be that species like L. jensenii are more easily cultured on blood plates than for example L. crispatus and therefore show a lower discrepancy between DNA-concentration and culture. In conclusion, when preparation of the standard curve in this study had been based on cultured cells, L. jensenii would have been overestimated relative to the other species.
L. crispatus and L. jensenii L. crispatus can be found in samples of all grades but the log 10 median concentration/ml (MC) of this species is much higher in grade I (8.8) compared to grades II (4.2) and III (5.2). Although the difference between the log 10 quantity of L. crispatus in grade Ia and grade Ib is not significantly different (p = 0.096), possibly because of the low sample size, a different profile is observed for these grades (Figure 2 and 3).
L. crispatus is present in high inoculum (i.e. at least) in all 8 grade Ia samples (log 10 MC 9.0), in most (i.e. 6/10) grade Iab samples (log 10 MC 8.9), but only in 3/12 grade Ib samples (log 10 MC = 5.6). Our study confirms that a grade Ia microflora is characterized by high concentrations of L. crispatus, whereas grades Iab and Ib consist of a more diverse microflora, with different Lactobacillus species present in comparable amounts.
Also, in grade Ib, high concentrations of L. crispatus can be found but on Gram stain especially long and small Lactobacillus cell types, which were suggested to be more typical for L. gasseri [24], are observed.
Based on cloning of the 16S rDNA library from the indigenous microbiota of three women with normal vaginal microflora, Verhelst et al. [14] found an almost pure population of L. jensenii in one of them and concluded that this species was associated with a normal vaginal microflora, but in this study (71 swabs) we can not correlate L. jensenii with either normal or disturbed vaginal microflora since it is present sporadically in samples of all grades, and in both high and low numbers. Its presence in 8/10 grade Iab samples, compared to 1/8 grade Ia samples is striking (p = 0.024).  [12], and in which G. vaginalis was shown to be associated with A. vaginae.

L. gasseri and L. iners
Overall L. gasseri and L. iners are abundantly present in most grades. Strikingly, L. gasseri is virtually absent in grade III whereas L. iners is virtually absent in grades Ia and II.
In our study we found a high concentration of L. iners to be clearly associated with low concentrations of L. gasseri, which is more prevalent in normal (grade Ia, grade Ib, grade Iab) and mildly disturbed flora (grade II). This was confirmed by Spearman rank testing, which showed that L. gasseri and L. iners are significantly negatively correlated to each other in grades II and III (r = -0.793, p < 0.0001), but also when all samples are considered together (r = -0.397, p = 0.001).
It can be noted that even the single grade III sample with a high concentration of L. gasseri was also one of the two grade III samples with low concentration of L. iners. In addition, we found a positive correlation between G. vaginalis and L. iners. The distribution frequencies over the different grades of L. iners and G. vaginalis are quite similar, i.e. present in most grades but predominantly in grade III, but different from A. vaginae, i.e. present almost exclusively in grade III, which may explain why L. iners does not correlate with A. vaginae. This distribution of L. iners was also observed in the T-RFLP study of Thies et al.
[32], with 32 out of 50 BV samples and 11 out of 20 normal microflora samples containing the L. iners TRF. The L. gasseri group TRF was observed only twice, in women with normal microflora. Unfortunately, these authors did not distinguish between grade II and III.
According to some research groups [35], L. iners is the most abundant vaginal Lactobacillus species, not only in black African women (64% of 241 healthy premenopausal Nigerian women) but also in white women from Sweden, the US and Canada and is considered as even more typical for normal vaginal microflora than L. crispatus. Although L. iners was present in high numbers in grades Iab and Ib, we found L. crispatus and L. gasseri to be more abundant in grade Ia. Ferris et al. [17] concluded that L. iners is a more transitional species, present in recently cured patients. They reported the predominance of L. iners in 5 patients with BV after metronidazole treatment, whereas for the sixth patient, the only one with a complete treatment failure, L. iners was present but not predominant. These results were confirmed by Jakobsson and Forsum [36].
Interestingly, it has been observed by the group of Taylor-Robinson that grade II microflora responds poorly to clindamycin treatment whereas most subjects with grade III microflora readily reverted to grade I [37,38] and also that women with clindamycin treated grade III microflora had a better pregnancy outcome than women with clindamycin treated grade II microflora [37]. In agreement, the predominance of clindamycin susceptible L. iners in grade III might explain the observations of Taylor-Robinson et al. [38].
Finally, in case grade II would be an intermediate stage in between transition from grade I to grade III, one would expect to see an increase in L. iners and a decrease in L. gasseri, since the former is present in high numbers in grade III and the latter almost absent, but we observe exactly the opposite, i.e. nearly complete absence of L. iners and high numbers of L. gasseri in grade II.
In order to establish whether this could be related to the differential presence of L. gasseri and L. iners in these grades, with L. gasseri predominant in grade II, we determined the MIC for clindamycin for 10 strains of each. We found that 8 out of 10 L. iners strains were inhibited by less than 0.25 μg/ml, whereas 9 out of 10 L. gasseri strains had a MIC value of 2 or more μg clindamycin/ml. Although L. gasseri cannot be considered definitely as the causative micro-organism responsible for the adverse pregnancy outcome that has been associated with grade II, our data indicate that it can be considered as contributing to the poor response to clindamycin treatment.
To test the hypothesis that L. gasseri and L. iners are mutually exclusive, e.g. by the production of bacteriocins, we carried out inhibition tests between both species. However, with the method we used we could not observe mutual inhibition between both species. BV. The discrepancy may be explained because we take also L. iners and L. gasseri into account.
To further resolve these discrepancies we carried out Gram staining of several strains of each L. gasseri and L. iners. It is of importance to note that L. iners has not only long escaped from our attention due to its inability to grow on MRS agar, but also hides as a Lactobacillus from Gram stains, because it stains Gram-negative and its cell morphology is rather coccobacillar than bacillar. Since it is the predominant Lactobacillus species in BV microflora -even present in high numbers -this has led to the false assumption that the BV microflora is devoid of lactobacilli. Grade I-like was described only recently by Verhelst et al.
[24] as a grade that, on Gram stain, resembles grade I because of the Gram positive rods, but with the use of molecular methods it was shown that these rods were Bifidobacterium spp. instead of Lactobacillus spp.
Again this study confirms the separate nature of grade Ilike vaginal microflora. A significantly lower concentration of L. crispatus (log 10 MC = 5.5 cells/ml) is present compared to grade I (log 10 MC = 8.8 cells/ml, p = 0.014) and much lower concentrations of G. vaginalis (log 10 MC = 4.0 cells/ml, p < 0.0001) and A. vaginae (log 10 MC = 1.6 cells/ml, p = 0.003) are present in grade I-like microflora compared to grade III (respectively log 10 MC = 9.2 cells/ml and log 10 MC = 9.0 cells/ml).
introitus. The first swab was used to prepare a smear on a glass slide for the purpose of grading as described by Verhelst et al. [14]. The second swab was returned to a sterile tube for the purpose of DNA extraction (dry swab). Both swabs were sent to the microbiology laboratory and were processed within 4 hours.
The study protocol was approved by the ethical committee of the Ghent University Hospital and individual written informed consent was obtained.  Table 3. Position of the primers on the 16S rRNA-gene is shown on Figure 1. Optimal annealing temperature of each primer set was established by gradient PCR and analysis on an agarose gel. In addition, already described primers for L. crispatus and L. gasseri [26] and G. vaginalis [27] were used. Primer sequences and cycling conditions are summarized in Table 4. Primer concentrations were 100 nM each except for the assays for G. vagi-nalis and L. iners for which the primer concentrations were 200 nM.

Construction of the standard curves for real-time PCR
To construct standard curves for the real-time PCR's, A. vaginae (CCUG 38953 T ), L. crispatus (PB2003/125-T1-1), L. gasseri (FB102-1) and L. jensenii (PB2003/204-T1-1) were cultured on TSA + 5% sheep blood (Becton Dickinson) and G. vaginalis (LMG 7832 T ) and L. iners (BVS11) were cultured on CNA agar (Becton Dickinson) + 5% human blood. A suspension was made in MRS Broth (Oxoid, Drongen, Belgium) and DNA was extracted. The DNA concentration of this stock was determined ten times by using the Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, USA) and the mean value was used for further calculations. For each strain a tenfold dilution series was prepared by dilution of the DNA stock in HPLC grade water. Dilutions were aliquoted and stored frozen at -20°C.

Real-time PCR
The qPCR Core Kit for SYBR Green I (Eurogentec) was applied and analysis was performed on the ABI 7300 realtime PCR system (Applied Biosystems, Foster City, CA).
Reactions were done in PCR mixtures containing 2.5 μl of DNA extract, 2.5 μl of 10 × Reaction Buffer, 3.5 mM MgCl 2 , 0.2 mM dNTP mixture, 0.625 U HotGoldStar Taq polymerase, 0.75 μl SYBR ® Green I, diluted 10-fold in DMSO and the appropriate primer concentration, adjusted with HPLC grade water to 25 μl. Primer concentrations are summarized in Table 4. Each run included a standard curve and each sample was run in triplicate. In case the result was not in the range of the standard curve, the samples were diluted tenfold and analyzed in triplicate again. The median log 10 cells/ml were expressed as per 1 ml elution buffer.