https://orcid.org/0000-0002-8407-7421
Figures
Abstract
Vaginal colonization with lactobacilli has been linked to the health of the lower urinary tract in women. There is growing evidence that the bladder has its microbiome related closely to the vagina. In this study, we compared the three common vaginal Lactobacillus species (L. jensenii, L. iners and L. crispatus) in vaginal and urine samples to identify factors that influence urinary detection and the quantity of Lactobacillus. We used quantitative real-time PCR (qPCR) assays to measure the concentration of Lactobacillus jensenii, L. iners and L. crispatus in paired vaginal swabs and clean-catch urine samples from pre-and post-menopausal women. We compared demographic variables and vaginal Lactobacillus quantity between women with vaginal detection of at least one of the three species, detection in both vagina and urine, or urine only. We performed Spearman correlation between vaginal and urinary quantities of each species. We used multivariable logistic regression models to determine predictors of detectable Lactobacillus species in both samples (vs. vagina only or urine only). Models were adjusted for variables selected a priori: age, BMI, condom use, and recent sexual activity. Ninety-three paired vaginal fluid, and urine samples were included in the final analysis. 44 (47%) had no detectable Lactobacillus species in their urine samples, and 49 (53%) had at least one of the three Lactobacillus species (L. jensenii, L. iners and L. crispatus) detected in urine. Most women were white (91.4%), with a mean age of 39.8 ±13.8 years. The two groups were similar in demographics, gynecologic history, sexual history, recent use of antibiotics or probiotics within 7 days of sample collection, Nugent scores, and urine-specific gravity. Among the three Lactobacillus species, L. jensenii was more commonly detected in urine than the other two. For all three species, detection in the urine sample alone was infrequent. The concentrations of all three species were higher in vaginal samples than in urine samples. For all three Lactobacillus spp., vaginal abundance was associated with the urinary abundance of the same species even after adjusting for the Nugent score. In Spearman correlation analysis, urinary and vaginal Lactobacillus concentrations were positively correlated within the same species, with the most significant correlation coefficient for L. jensenii (R = 0.43, p<0.0001). Vaginal quantities were positively correlated between the three species, as were urinary quantities to a lesser extent. There was no meaningful correlation between the urinary quantity of one Lactobacillus sp. and the vaginal quantity of another species. In summary, the vaginal quantity of Lactobacillus was the most significant predictor of concurrent detection of the same species in the bladder, confirming the close relationship between these environments. Strategies to promote vaginal Lactobacillus colonization may also bring urinary colonization and the health of the lower urinary tract.
Citation: Kim Y, Bergerat-Thompson A, Mitchell C (2023) Vaginal and urinary evaluation of lactobacilli quantification by qPCR: Identifying factors that influence urinary detection and the quantity of Lactobacillus. PLoS ONE 18(4): e0283215. https://doi.org/10.1371/journal.pone.0283215
Editor: Brenda A. Wilson, University of Illinois Urbana-Champaign, UNITED STATES
Received: September 30, 2022; Accepted: March 3, 2023; Published: April 14, 2023
Copyright: © 2023 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors received no specific funding for this work.
Competing interests: No authors have competing interests
Introduction
Analysis of urine using molecular methods and enhanced culture techniques has identified a commensal community of bacteria in the bladder, an area once presumed to be sterile [1–3]. Bladder microbiota have been associated with a range of lower urinary tract disorders in women, including overactive bladder [4, 5], urgency and stress urinary incontinence [3, 6–8], painful bladder syndrome [9], and post-operative urinary tract infections [10]. Identifying determinants of bladder microbiota may offer opportunities to improve treatment for these conditions.
Urinary microbial communities differ in composition between men and women, suggesting that differences in anatomy impact the seeding of the bladder with bacteria [2]. The vaginal microbiome seems a likely source of female urinary microbiota. Among pre-menopausal women, the relative abundance of Lactobacillus, Gardnerella, Prevotella, Ureaplasma, and Escherichia species were correlated between vaginal and mid-stream voided urine samples [11]. In postmenopausal women, an expanded culture of catheter-collected urine samples demonstrated increased urinary Lactobacillus abundance after vaginal estrogen treatment [12].
Comparisons of the relative abundance of microbes in vaginal and urine samples using 16S rRNA sequencing have suggested a significant concordance between communities in the vagina and urine [11, 13]. This correlation was largely due to the high relative abundance of Lactobacillus in both vagina and the urine samples [11, 13]. However, this type of sequencing only measures relative quantities and may not detect lower abundance members of the community. Absolute measurement of individual species through qPCR offers a different assessment of the impact of a given species: a 20% relative abundance in a community of a thousand organisms may have a very different biological impact than 2% of a million organisms. In this study, we compared the absolute abundance of three common Lactobacillus species (L. jensenii, L. iners and L. crispatus) in vaginal and urine samples to identify the factors influencing urinary detection and the quantity of Lactobacillus.
Materials and methods
Study setting, population, and design
Women presenting for ambulatory gynecological care at Massachusetts General Hospital were recruited for an observational cohort study of vulvovaginal symptoms and provided informed consent (IRB # 2014P001066) between August 2014 and May 2017. Women were excluded if their age was under 21 years, if they were pregnant, had known HIV infection or immunosuppression due to illness or medication use, or were prisoners. All racial and ethnic groups were included.
Participants completed questionnaires about general health history, sexual history, recent antibiotics, vaginal product use, and the presence of vulvovaginal symptoms. All participants underwent pelvic examination and obtained a vaginal swab sample and a midstream clean catch urine sample. Paired vaginal and urine samples from 100 subjects were analyzed by quantitative PCR (qPCR) for the presence and abundance of three Lactobacillus species: L. jensenii, L. iners and L. crispatus.
Sample collection and storage
Urine samples were stored at 4°C for < 24 hours and aliquoted into 1.8 mL cryovials for storage at −80°C until thawed for analysis. Vaginal swabs were stored at –80°C until analysis. Gram-stained slides of vaginal fluid underwent evaluation using the Nugent criteria [14].
Bacterial DNA extraction of vaginal and urine samples
Vaginal swabs were thawed at room temperature, re-suspended in 450μLof phosphate-buffered saline solution, then vigorously vortexed for 2 minutes, and the swab was removed [15]. To prevent crystallization in urine samples, after thawing at 4 C, Tris-EDTA (1M) was added at 10% of urine volume before collecting the sediments [16]. DNA from the vaginal swab and urine samples was extracted using DNeasy UltraClean Microbial Kit (QIAamp).
Quantification of Lactobacillus spp. by quantitative real-time PCR (qPCR)
The QuantStudioTM 7 Flex System (Thermo Fisher Scientific) and Sequence Detection Software version 1.3 (Applied Biosystems) were used to perform and analyze all qPCR results. Species-specific primers sets, TaqMan probes, and thermocycler conditions are detailed in S1 Table. Each reaction was performed in a final volume of 20μL containing 10μL GoTaq® Master Mix 2X (Promega), 0.8μL of each primer, 3μL nuclease-free water, and 2μL template DNA as previously described [17]. Results are reported as 16S rRNA gene copies/swab, which were calculated using a plasmid standard curve ranging from 2.5 to 106 copies/reaction. Each assay was run with water-blank control samples to assess for contamination.
The average values were used to calculate the final concentration per sample (i.e., per vaginal swab sample or per mL of urine). Samples detected below the lower limit of quantification (2.5 gene copies/reaction) or undetected were assigned the value of 0.5 copies/sample for logarithmic transformation. When none of the three Lactobacillus species were detected in either vagina or urine, the participant was excluded from the analysis.
Statistics
Variables were compared between groups using an independent t-test (continuous), a Chi-square test, and Fisher’s exact test (categorical). A linear regression was performed to assess the association between vaginal and urinary quantities of each species. Because the Nugent score is a rough approximation of the relative abundance of Lactobacillus vs. other bacterial morphotypes, we adjusted this analysis for the Nugent score to examine how an absolute quantity of vaginal lactobacilli impacts urinary quantity at a similar relative abundance. A multivariable logistic regression model was used to determine variables associated with the urinary detection of each Lactobacillus species. The model included the a priori selected variables which we hypothesized might impact urinary colonization with microbes: age, BMI, condom use, vaginal cleansing history, and vaginal Lactobacillus quantity. Age and menopausal status are highly correlated; thus, we only included age in the model. Spearman correlation was used to evaluate associations between vaginal and urinary quantities of all Lactobacillus spp. using log10-transformed 16S rRNA gene copy numbers. All analyses were conducted in Stata (16.1, StataCorp LLC, College Station, TX) and GraphPad Prism version 9 (GraphPad Software, Inc). A p-value <0.05 was considered statistically significant.
Results
Study population
Paired urine and vaginal samples were available for analysis from 100 women enrolled between August 2014 and May 2017. DNA was successfully extracted and analyzed from all specimens.
Ninety-three paired samples had at least one of the targeted Lactobacillus species (L. jensenii, L. iners and L. crispatus) detected in either vaginal or urine samples. Seven paired samples with no detectable quantities of any of the three Lactobacillus species in either sample were excluded from subsequent analyses. Of the remaining 93, 44 (47%) had no detectable Lactobacillus species in their urine samples, and 49 (53%) had at least one of the three Lactobacillus species (L. jensenii, L. iners and L. crispatus) detected in urine (Fig 1). The two groups were similar in demographics, gynecologic history, sexual history, recent use of antibiotics or probiotics within 7 days of sample collection, Nugent scores, and urine-specific gravity (Table 1). People with none of the three species of lactobacilli detected in urine had a higher BMI (p = 0.016)
Paired vaginal swabs and clean catch urine from 100 participants were assessed with species-specific qPCR for these three species of lactobacilli. Seven samples with no detection of any of the target species were excluded from the analysis.
Among the three Lactobacillus species, L. jensenii was more commonly detected in urine than the other two species (Fig 1). For all three species, detection in the urine sample alone was infrequent. The concentrations of all three species were higher in vaginal samples than in urine samples (Fig 2; S1 Fig) (p<0.001).
Only pairs with the detection of the Lactobacillus species in the urine sample are depicted.
Predictors of urinary detection of Lactobacillus
All three Lactobacillus spp., vaginal abundance was associated with the urinary abundance of the same species even after adjusting for the Nugent score (Table 2). When only the samples with quantifiable urinary lactobacilli were included in the analysis, the relationship remained true for L. jensenii only (S2 Table). Because there was a high proportion of urinary samples with undetectable or unquantifiable levels of Lactobacillus spp. in the urine, we created a binary categorical variable for the presence of each species in the urine sample. In multivariable logistic regression analysis, the detection of Lactobacillus spp. in urine samples was significantly associated with the quantity of each Lactobacillus spp. in vaginal samples after adjusting for age, BMI, condom use, and vaginal cleansing history (Table 3). False detection rates were 13.56% (L.jensenii), 13.70% (L. iners) and 16.67% (L. crispatus) when we used the probability threshold that maximizes Youden’s J Index (Table 3). For L. iners, a history of recent condom use was also significantly associated with detecting that species in urine (Table 3). Additional chi-square analysis comparing binary presence/absence of each species in both urine and vaginal samples showed that while vaginal detection of L. jensenii was associated with urinary detection (p<0.001), this was not the case for L. iners and L crispatus (p>0.05) (S3 Table).
Correlation of Lactobacillus quantity between paired vaginal and urine samples
In Spearman correlation analysis, urinary and vaginal Lactobacillus concentrations were positively correlated within the same species, with the most significant correlation coefficient for L. jensenii (Fig 3. R = 0.43, p<0.0001). Vaginal quantities were positively correlated between the three species, as were urinary quantities to a lesser extent. There was no meaningful correlation between the urinary quantity of one Lactobacillus sp. and the vaginal quantity of another species.
Each box is shaded according to the value of the Spearman correlation coefficient, and numbers represent the p-value.
Discussion
In this study, we demonstrate a significant association between the vaginal quantity of three different Lactobacillus species and the detection of that species in a clean-catch urine sample. Using species-specific, quantitative PCR, we showed a correlation between vaginal and urinary quantities of the three species of lactobacilli: L. jensenii, L. iners and L. crispatus. This finding not only supports the growing evidence that there is an inter-relatedness between vaginal and urinary microbiomes but further reveals that the association is likely related explicitly to the absolute abundance of each Lactobacillus species.
Comparative studies of relative abundance using 16s rRNA sequencing and expanded quantitative urine and vaginal cultures have previously demonstrated significant overlap between vaginal and urinary microbiomes [3, 7, 11, 18]. Although the two methods are different, both identify a close association between the two microbiomes, primarily due to the predominance of the genus Lactobacillus[3, 7, 11, 18]. Specifically, the Lactobacillus species that showed the closest vaginal-urine correlation were L. jensenii, L. iners and L. crispatus [11]. Our use of quantitative PCR allowed us to demonstrate that the absolute amount of vaginal Lactobacillus spp., not just the relative amount, is an essential determinant of urinary colonization by the same species. Thus, even if there is a disruption to the microenvironment of vaginal flora, which causes a lowering of the relative abundance of lactobacilli, an individual may still have urinary colonization of the same species if the absolute abundance is sufficient.
In a recent study using 16s rRNA sequencing, the correlation between the relative abundances of vaginal and urinary Lactobacillus was similar between people with mixed urinary incontinence and a healthy control group [11]. The existence of this close relationship between urinary and vaginal Lactobacillus colonization, even in the control group, is additional evidence that many bacteria are likely to be shared between the vagina and bladder, not just by pathogenic organisms like E. coli. Whole genome sequencing of bacterial isolates from the vagina and bladder in 77 individuals revealed that strains of L. iners and L. crispatus isolated from an individual were the same in the two samples [18]. This is congruent with our finding that the effect of the vaginal abundance of Lactobacillus species on urinary abundance is species-specific. The abundance of one vaginal Lactobacillus species did not affect the abundance of other evaluated species in the urine. Knowing that Lactobacillus species have a diverse impact on lower urinary tract health, this species-specific association may play an essential role in creating the most effective treatment strategies.
Clinically, studies involving individuals with and without lower urinary tract symptoms have indicated that disruption of the genitourinary microbiome likely contributes to the process of the pathogenesis of many symptoms [7, 19, 20]. A health-associated microbiome is characterized by decreased urinary bacterial diversity (price bladder, Adebayo), which usually means dominance by the genus Lactobacillus. Differences in the relative abundance of Lactobacillus species are associated with various lower urinary tract disorders (3,8,11,19). In a subgroup analysis of women younger than 51 years old, 16s rRNA sequencing of urine and vaginal samples showed that the control group had a higher Lactobacillus relative abundance [11]. The study recognized its limitations of having reported the associations mostly at the genus level. Our study, which demonstrated the association at the species level with the aforementioned qPCR method, demonstrated that increasing the vaginal quantity of Lactobacillus species increased the likelihood of detecting the same species in the urine.
In preserving vaginal health, the importance of symbiosis of H2O2-producing Lactobacillus species is well recognized [7, 21–23]. Maintaining a Lactobacillus-dominant vaginal microbiome is associated with lowered risks of sexually transmitted infections and vulvovaginal disorders, such as bacterial vaginosis and candidiasis, and improved obstetrical outcomes [21–25]. Similarly, microbiome studies of the bladder show an essential function of Lactobacillus species. For example, L. crispatus, universally accepted as health-promoting vaginal Lactobacillus, has also been established as an essential organism associated with urinary health, such as lowering the risks of urinary tract infections and urinary incontinence [3, 8, 12].
As mentioned above, our results strongly suggest species-specific associations between vaginal and urinary colonization, which implies that to promote urinary colonization with specific species, its vaginal quantity must be increased. This likely applies to other bacteria found in the genitourinary system as well. Exploring the species-specific effects of various bacteria in the bladder is essential. In a culture-based study, urinary L. gasseri was more commonly found in women with urinary urge incontinence [26]. In another study, Atopobium vaginae were associated with painful bladder syndrome [27]. Future species-specific studies involving a larger group of matched samples with and without lower urinary tract symptoms are warranted to provide further insight into developing effective preventative and curative therapies.
Although we did not find age or menopausal status to be an independent factor for urinary detection of three studied Lactobacillus spp., this is likely due to a lack of power, given the relatively narrow age range of participants, and the small number of postmenopausal participants. Aging has been shown to be a predictor of increased urinary microbiome diversity in several studies [20, 28]. Recently, a longitudinal study of 12 weeks of use of vaginal estrogen treatment in postmenopausal patients demonstrated a significant increase in urinary Lactobacillus spp. using the enhanced urine culture technique [12]. Although the study did not demonstrate increased vaginal quantities of Lactobacillus during the study period, it did show a significant decrease in the diversity within the microbial community, consistent with greater Lactobacillus dominance of vaginal communities [21, 24, 25, 29, 30]. Other studies with more extended follow-up periods have shown increased vaginal Lactobacillus with vaginal estrogen administration [29, 31], which likely contributes to more significant urinary Lactobacillus colonization and decreases UTI in women using topical estrogen preparations [32–34].
A major limitation of our study was the small number of relatively homogenous participants recruited for this study. Additionally, we did not have data on urinary symptoms from validated questionnaires, and we could not correlate and control for the presence of lower urinary tract disorders. Our study used mid-stream, clean catch samples rather than urine collected with a catheter; thus, some contribution of contamination during collection cannot be ruled out. However, the fact that we found urinary lactobacilli in people with no vaginal lactobacilli detected, and that associations were species-specific suggests that this was not a significant issue.
Conclusion
Our results add to data suggesting that increasing the absolute quantity of vaginal lactobacilli will likely promote the prevalence of urinary Lactobacillus colonization. Additionally, our findings suggest vaginal colonization leads to direct seeding of the bladder since vaginal quantities were only correlated with quantities of the same species in the urine. Thus, any probiotic products for urinary health should contain species that will colonize the vagina.
Supporting information
S1 Fig. Quantity of L. jensenii, L iners or L. crispatus detected in paired vaginal and urinary samples by species-specific qPCR.
This figure includes samples without detectable Lactobacillus species in the urine sample.
https://doi.org/10.1371/journal.pone.0283215.s001
(TIF)
S1 Table. Primers, probe sequences, and PCR conditions for TaqMan assays.
https://doi.org/10.1371/journal.pone.0283215.s002
(DOCX)
S2 Table. Association between vaginal and urinary 16S rRNA gene copy numbers of L. jensenii, L iners and L. crispatus, excluding samples without quantifiable detection in the urine.
https://doi.org/10.1371/journal.pone.0283215.s003
(DOCX)
S3 Table. Association between vaginal and urinary detection of the same Lactobacillus species (N = 93).
https://doi.org/10.1371/journal.pone.0283215.s004
(DOCX)
Acknowledgments
Presentation: This submission was presented as a short oral at the 2021 American Urogynecologic Society meeting on October 15, 2021, in Phoenix, Arizona.
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