Lactobacillus iners and genital health: molecular clues to an enigmatic vaginal species

Abstract

Purpose of review

Vaginal lactobacilli are recognized as important drivers of genital health including protection against bacterial vaginosis and sexually transmitted infections. Lactobacillus iners is distinct from L. crispatus, L. gasseri, and L. jensenii by its high global prevalence in vaginal microbiomes, relatively small genome, production of only L-lactic acid, and inconsistent associations with genital health outcomes. In this review, we summarize our current understanding of the role of L. iners in the vaginal microbiome, highlight the importance of strain-level consideration for this species, and explain that while marker gene-based characterization of the composition of the vaginal microbiota does not capture strain-level resolution, whole metagenome sequencing can aid in expanding our understanding of this species in genital health.

Recent findings

L. iners exists in the vaginal microbiome as a unique combination of strains. The functional repertoires of these strain combinations are likely wide and contribute to the survival of this species in a variety of vaginal microenvironments. In published studies to date, strain-specific effects are aggregated and may yield imprecise estimates of risk associated with this species.

Summary

The worldwide high prevalence of Lactobacillus iners warrants more research into its functional roles in the vaginal microbiome and how it may directly impact susceptibility to infections. By incorporating strain-level resolution into future research endeavors, we may begin to appreciate L. iners more thoroughly and identify novel therapeutic targets for a variety of genital health challenges.

Introduction

Lactobacillus iners is the most prevalent member of the vaginal microbiome, and yet, its function in the complex vaginal microenvironment remains incompletely understood. The role of L. iners has been a topic of debate (see reviews by Petrova et al., Vaneechoutte et al., Zheng et al., and Carter et al.) due to its unique characteristics among vaginal lactobacilli, which implicate it to differing degrees in a variety of adverse genital health outcomes [1–4]. It appears a L. iners-dominated vaginal microbiota may be less optimal than those predominated by Lactobacillus crispatus, L. jensenii, or L. gasseri, but L. iners dominance appears to remain more protective compared to Lactobacillus-deficient microbiota containing bacterial vaginosis (BV) associated bacteria, including Gardnerella, “Ca. Lachnocurva vaginae”, and other strict and facultative anaerobes. However, there is evidence that this generalization about L. iners is overly broad and does not hold for all populations and outcomes. Research to better understand the functional roles that L. iners plays in the vaginal microbiome is active and ongoing. In this review, we highlight recent ecological and ‘omics-based findings that advance our understanding of the diversity within L. iners. We argue that L. iners should be more precisely characterized and more rigorously evaluated if we are to understand its contributions to genital health.

The Vaginal Microbiome and its Most Prevalent Member, Lactobacillus iners

The vaginal microbiome is a critical contributor to genital and reproductive health as it presents a first line of defense against cervicovaginal infections [5–20]. This is achieved in part through the influential action of Lactobacillus spp., including L. crispatus, L. gasseri, L. jensenii and L. iners. Vaginal lactobacilli provide broad-spectrum protection against a range of pathogens through their production of copious amounts of lactic acid and bacteriocins [21–31].

Globally, L. iners is the most prevalent vaginal species in reproductive-aged women across the world as noted by studies conducted in North and South America, Europe and the UK, Sub-Saharan Africa, East and South Asia, and Australia, and it is commonly detected in samples from peri- and post-menopausal women [22, 32–54]. L. iners is observed in vaginal environments with both high (>4.5) and low (<4.5) vaginal pH and can exist alone or co-reside with other vaginal Lactobacillus spp. or anaerobic bacterial species [34, 35].

Vaginal lactobacilli are not Equally Protective Against Pathogens

L. iners may not provide equivalent protection against cervicovaginal infections compared to other vaginal lactobacilli. One reason is related to lactic acid production: while acidification of the vaginal environment is one protective mechanism of lactic acid, there is evidence that lactic acid isomers can uniquely contribute to protection against vaginal pathogens [21, 24, 55–57]. Lactic acid exists as two isomers, L(+) and D(−) lactic acid, and both reduce vaginal pH [58]. L-lactic acid originates from both the vaginal epithelia (minor contribution to total lactic acid concentration) and vaginal Lactobacillus spp., while D-lactic acid is produced by L. crispatus, L. gasseri, and L. jensenii, but not L. iners [59–63]. A small study of 31 participants indicated that while L-lactic acid concentrations did not substantially differ, D-lactic acid concentrations were higher among L. crispatus-dominated microbiota than L. iners dominated microbiota. While both isomers show protective properties, in vitro assays demonstrate D-lactic acid appears to contribute to greater protection against Chlamydia trachomatis (CT) infection and alters the diffusional properties of cervicovaginal mucus to increase trapping of HIV-1 pseudoviruses [21, 25]. In contrast, L-lactic acid may have more potent stereochemical-dependent virucidal activity against HIV than D-lactic acid, potentially by triggering viral protein unfolding [24].

Compared to L. crispatus-dominated vaginal microbiota, L. iners-dominated microbiota have been associated with increases in BV incidence, HIV acquisition, and CT incidence and prevalence [40, 64–69]. A recent meta-analysis of six studies found 3.4-fold higher odds of CT detection among individuals with L. iners-dominance compared to those with L. crispatus-dominance (95% CI 2.1 – 5.4) [4]. L. iners-dominated microbiota may also be more likely to harbor Candida albicans, though conflicting results exist [70–73]. In a study from McKloud et al., higher relative abundances of L. iners were observed in samples from patients with recurrent vulvovaginal candidiasis (RVVC) compared to healthy individuals [71]. On Candida albicans detection by PCR or microscopy, two studies reported associations between Candida and L. iners-dominated microbiota [70, 72], while a significant association was not observed in another [73]. While there is recent data on associations between cervical human papillomavirus (HPV) infections and BV-associated vaginal microbiota, there does not appear to be an association between L. iners and cervical HPV detection in either cross-sectional or prospective longitudinal studies [74–77]. While some L. iners dominated microbiota exhibit temporal stability, the relative increases in the permissive nature of L. iners to infections may be related to the reduced stability and greater tendency to fluctuate into Lactobacillus-deficient states compared to L. crispatus dominance [78, 79]. These fluctuations may result from endogenous or exogenous pressures (i.e., hormonal changes due to menstrual cycle or contraception, sex, intravaginal practices, etc.) which serve to open ecological niches and provide opportunities for other species or pathogens to expand or invade the community [80]. However, it is unknown why some L. iners dominated microbiota are more resilient to these pressures than others.

Clinically, in studies of BV antibiotic treatment efficacy, recent evidence suggests that the presence of L. iners prior to treatment may be related to BV recurrence. In two independent cohorts, Lee et al. showed that higher pre-treatment ratios of L. iners to Gardnerella vaginalis were associated with BV recurrence [80]. They validated these findings in vitro using L. iners type strain ATCC 55195 and hypothesized that L. iners may contribute to post-treatment BV recurrence via metronidazole sequestration, which was supported by mathematical modeling. Mollin et al. observed that pre-treatment relative abundances of L. iners trended higher in patients with refractory Amsel-BV compared to those in remission or with recurrent Amsel-BV, though this was not statistically significant [82]. Serebrenik et al. also found no significant effect of pre-treatment L. iners on BV treatment failure [83]. In addition, following BV treatment, L. iners is often the most abundant species, which is hypothesized to be due in part to its continued presence from BV diagnosis through treatment and/or its ability to emerge faster than other species after BV treatment [82–89]. Together, these findings suggest L. iners may play important roles in the events surrounding the incidence and treatment of BV, but more work is needed.

Detection of Lactobacillus iners Misses the Mark

To date, most studies evaluate the vaginal microbiota using morphological or marker gene-based assays such as amplicon sequencing of the 16S rRNA gene. Unlike other vaginal lactobacilli, L. iners has a thin peptidoglycan layer in its cell wall contributing to its variable morphology in Gram stained vaginal smears [88]. It can appear at times as gram-positive or gram-variable and as bacilli or cocci [90, 91]. Variation in cell morphology is also observed within and between isolated strains of L. iners (Fig. 1). Though there are multiple approaches to identifying BV, one most often used in the context of research studies, the Nugent score, is reliant upon Gram stained vaginal smears (now termed Nugent-BV) which is dependent on the amount of gram-positive bacilli and gram-negative cocci [92, 93]. L. iners’ morphological plasticity is problematic because it may cause misidentification of L. iners as Gardnerella morphotypes and subsequent misclassification of non-Nugent-BV samples as Nugent-BV [94]. As a result, in some cases, L. iners may be spuriously linked to BV.

Fig. 1.

Gram stains of L. iners cultured isolates highlight variable cell morphology and Gram staining within (arrows) and between strains.

Marker gene sequencing methods have improved on morphological characterizations of the vaginal microbiota by providing species-level identification of major vaginal bacteria [55]. However, recent work with whole genome metagenomic sequencing indicates that vaginal microbiomes are comprised of populations of multiple strains of the same species [95]. Unfortunately, marker gene sequencing is incapable of providing the resolution necessary to characterize the subtle differences that exist between L. iners strains (i.e., functional information) and between L. iners-predominated microbiomes. For example, the 5–6 copies of the 16S rRNA gene are not identical within a strain genome (Fig. 2).

Fig. 2.

Single nucleotide variants (SNV) in Lactobacillus iners 16S rRNA genes (indicated with “v”) are not specific to a genomically-distinct L. iners strain. (“-“ indicates spaces in the alignment). Strain names indicated by colored font.

Furthermore, the same single nucleotide variants (SNVs) are observed in genomically-distinct strains (Fig. 2: see C0210C1 and C001D1 or C0094A1 and C0322A1). While some SNVs in the 16S rRNA gene may be specific enough to a given strain that they reliably identify the strain (e.g., C0059G1), there are too few complete genomes of L. iners strains available to be certain. In addition, amplicon sequence variants, which have been associated with health outcomes, could represent combinations of strains [96]. However, caution should be taken when interpreting these associations because amplicon sequence variants are prone to contamination [97]. Regardless of whether data are operationalized as amplicon sequence variants or operational taxonomic units, 16S rRNA gene amplicon sequencing aggregates functionally diverse L. iners strains into a single species-level taxon. By aggregating distinct strains, associations estimated from marker gene sequencing data reflect a mixture of strain-specific associations and therefore may obscure true strain-level effects. We argue that the potentially different functional roles played by L. iners in the vaginal microbiome are poorly understood largely due to technology that has not provided the resolution needed to accurately evaluate the genomic diversity within this species.

The Importance of Strains in Understanding the Roles of Lactobacillus iners

L. iners has the smallest genome of vaginal lactobacilli but is estimated to have more genetic variation between its strains than other vaginal Lactobacillus spp. [62, 95, 98, 99]. Maintaining multiple strains with high genetic variation in a microbiome could support L. iners’ survival in the dynamic vaginal environment: while a portion of L. iners strains could be better adapted to co-exist with L. crispatus, others may be better equipped to compete against anaerobes in a BV-like state with higher pH. Similarly, some L. iners strains could predominate the microbiome largely alone. Each vaginal microbiome contains a distinct combination of strains [95]. However, these unique combinations cannot be captured using marker gene sequencing methods (Fig. 2). Instead, metagenomic sequencing provides a means to detect these combinations of L. iners strains, referred to as metagenomic subspecies [95]. Metagenomic subspecies are distinct combinations of strains of a species as defined by their genetic inventory, and these differences likely confer functional differences in the vaginal microbiome [95]. Importantly, each L. iners metagenomic subspecies present in the vaginal microbiota may have significant implications for adverse outcomes, such as the development of BV and altered susceptibility to STIs. For example, in a ten week study examining daily microbiota dynamics, participants with L. iners dominated microbiota throughout the majority of follow-up transitioned to molecular BV in 11% (n=85) of consecutive day-to-day comparisons [64]. This importantly highlights that the remaining 89% (n=722) of day-to-day comparisons among these participants did not result in BV. We posit that this may be due to specific L. iners metagenomic subspecies comprising the vaginal microbiomes and that certain metagenomic subspecies are more prone to transitioning to BV.

In vitro experiments of L. iners also require strain-level consideration. Often, in vitro studies utilize one or a few strains of L. iners and generalize findings to the entire species. For example, in one report, L. iners induced a significant proinflammatory response like Fannyhessea vaginae, while in another it was cited as having only little to no proinflammatory response [9100, 101]. The strain used in the former study was ATCC 55195 which originated from a patient with BV, and the latter study used CCUG 28746T, isolated from a healthy individual [102]. While experimental discrepancies likely contribute to the contradictory results, variations in gene content and expression between the two L. iners strains studied likely explain a greater degree of the differences in findings. An exploration of the strains’ genome sequences would be necessary to identify the potential functional features responsible for the differing phenotypes, but neither strain has complete genome sequences available. Currently, a major gap in understanding L. iners’ functional roles in the vaginal microbiome is the lack of a large collection of L. iners isolates with complete genomes available along with experimental phenotypic data and information about their environment of origin.

The Transcriptome of Lactobacillus iners: Another Layer of Complexity

Gene expression studies of the vaginal microbiome provide a glimpse of the multi-faceted roles played by L. iners. Tuning gene expression in response to environmental changes is an energy-saving survival strategy useful in dynamic environments, including the vagina [103]. One example in L. iners is the production of inerolysin (INY), a cholesterol-dependent cytolysin (CDC) with hemolytic and pore-forming capabilities [104]. The expression of INY can be greater in BV-like communities relative to Lactobacillus-predominated communities which could be linked to genetic diversity in strains of L. iners or that INY expression varies depending on the context of the composition of the microbiota [105, 106]. Another example is the expression of rodZ which controls cell shape. France et al. found rodZ expression increases when L. iners is present with L. jensenii, but not with BV-associated bacteria suggesting that cell morphology may reflect L. iners’ response to the surrounding microbiota (or their metabolic products) [106].

Lactobacillus iners as a Target for Improving Genital Health Outcomes

Given the high prevalence of L. iners in vaginal microbiomes, its presence in a range of vaginal environmental states, and its functional plasticity (whether through genetic diversity, maintenance of multiple strains, differential gene expression, or both), specific L. iners strains, genes, or products may be widely applicable targets for therapeutic or preventative modulation of vaginal microbiota. In their report on cysteine dependence of vaginal lactobacilli, Bloom et al. demonstrated that L. iners lacks transport systems for exogenous cysteine uptake, which are present in L. crispatus, L. gasseri, and L. jensenii [107]. L. iners instead relies on uptake of exogenous L-cystine, which is then converted to cysteine intracellularly [107]. In in vitro assays of mock BV-like communities, treatment with cystine uptake inhibitors and metronidazole promoted growth of L. crispatus, whereas treatment with metronidazole alone resulted in expansion of L. iners [107]. Thus, cystine uptake inhibitors or other agents that specifically inhibit L. iners may in the future complement BV treatment regimens to modulate microbiota composition in favor of non-iners Lactobacillus spp. expansion [107].

In general, L. crispatus has been associated with optimal vaginal health partly due to its global prevalence and associations with positive health outcomes [78]. However, L. crispatus predominance may not be a state that is reasonably attainable for some individuals due to host physiology or immunology. There do exist stable L. iners-predominated microbiota which may have a specific strain-level signature, i.e., metagenomic subspecies and could be optimal for some individuals [78]. A novel intervention may be modulation of unstable L. iners microbiota, which are associated with increased risk for BV, to a stable microbiota comprised of L. iners strains resistant to shifts.

Looking Forward

What must be done to further understand the role of L. iners in the vaginal microbiome and reveal its potential for contributing to genital health outcomes? First, progress must be made to isolate and characterize L. iners strains from a worldwide population. This collection must include representative genomes, the composition of the associated microbiota, host phenotype information (at least health or disease), and, ideally, geographic isolation data. From this resource, biochemical, genomic, and transcriptomic comparisons will reveal the range of strain variation that exists within L. iners. Second, because species exist as multiple strains in a single vaginal microbiome, metagenomic sequencing can facilitate large-scale functional characterizations of L. iners metagenomic subspecies and identify how metagenomic subspecies differ according to disease status [95]. Third, metatranscriptomic sequencing will reveal how L. iners metagenomic subspecies respond to the dynamic vaginal microenvironment and exogeneous pressures (e.g., antibiotic treatment, sexual activity, intravaginal practices). Ideally, these large-scale studies will be longitudinal and include patients’ clinical and demographic data and observe within-individual changes over time. Importantly, more bioinformatic tools must be developed to measure these aspects of the vaginal microbiome quickly, easily, and robustly. Finally, hypothesized mechanisms generated from these large-scale studies would warrant in vitro, and ultimately in vivo confirmation.

In summary, vaginal L. iners is highly prevalent in populations around the world, and therefore reconciling its role in the vaginal microbiome is urgently needed. In combination with clinical research, ‘omics technologies and large-scale cultivation efforts can provide opportunities to better investigate the varied roles L. iners plays in the vaginal microbiome and its contributions to genital health. Investigations of L. iners strains may identify promising novel treatment targets and prevention strategies that could directly contribute to improving genital health for diverse populations worldwide.