Article Text

Characteristics associated with Lactobacillus iners-dominated vaginal microbiota
  1. Juliano Novak1,
  2. Jacques Ravel2,
  3. Bing Ma3,
  4. Carolina Sanitá Tafner Ferreira1,
  5. Andrea da Rocha Tristão4,
  6. Marcia Guimaraes Silva1,
  7. Camila Marconi1,5
  1. 1 Department of Pathology, São Paulo State University (Unesp), Medical School, Botucatu, São Paulo, Brazil
  2. 2 Department of Microbiology & Immunology, University of Maryland Baltimore, Baltimore, Maryland, USA
  3. 3 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA
  4. 4 Department of Gynecology and Obstetrics, São Paulo State University (Unesp), Medical School, Botucatu, São Paulo, Brazil
  5. 5 Department of Basic Pathology, Federal University of Parana, Curitiba, Brazil
  1. Correspondence to Dr Juliano Novak, Department of Pathology, Universidade Estadual Paulista Júlio de Mesquita Filho Câmpus de Botucatu Faculdade de Medicina, Botucatu 18618-970, Brazil; juliano.novak{at}


Objective The protective role of Lactobacillus iners in the vaginal microbiota has been questioned. Recent studies have shown that L. iners is the dominating taxon in a large subset of women worldwide. The aim of this study was to identify sociodemographic, behavioural and clinical variables associated with L. iners-dominated community state type (CST) III in Brazilian women of reproductive age.

Participants and methods This study leveraged microbiota compositional data generated by sequencing of the V3-V4 16S rRNA gene from vaginal samples collected from 442 participants enrolled in a previous cross-sectional study that included 609 women in five geographical regions of Brazil. A total of 167 (27.4%) participants were excluded from the current study as they did not present a Lactobacillus-dominated vaginal microbiota. Data on sociodemographic and behavioural characteristics of the study population were obtained through face-to-face interviews. Participants were assigned to two study groups: those with L. iners-dominated CST III (n=222) and those with three distinct CSTs (I, II or V) dominated by another Lactobacillus spp. (n=220). Logistic regression analysis using a stepwise method was performed to test association between CST III and participants’ characteristics, considering their OR and 95% CIs.

Results Among the population characteristics assessed, L. iners-dominated CST III was independently associated with having two or more sexual partners (OR 3.27; 95% CI 1.50 to 7.11) and microscopic detection of Candida sp. on vaginal smears (OR 2.24; 95% CI 1.02 to 4.89). Other characteristics were inversely associated with CST III, including condom use (OR 0.59; 95% CI 0.38 to 0.91), higher educational level (OR 0.61; 95% CI 0.41 to 0.91) and diet containing milk/dairy intake (OR 0.43; 95% CI 0.20 to 0.90).

Conclusion Unprotected sex practices, number of sexual partners and lower educational levels may be useful for identifying women with L. iners-dominated microbiota and its suboptimal protective properties. L. iners microbiota does not seem to provide optimal protection against Candida sp. colonisation, warranting further investigation.

  • bacterial infections
  • vaginosis
  • bacterial
  • molecular biology
  • vaginal smears
  • sexual behaviour

Data availability statement

No data are available. Not available.

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Previous studies have shown that there are different types of vaginal microbiota in women of reproductive age, and they differ in terms of species composition and dynamics.1–3 At least five major types of vaginal microbiota or community state types (CSTs) have been identified.1 Four of these CSTs are dominated by one of four species of Lactobacillus (CST I: L. crispatus; CST II: L. gasseri; CST III: L. iners, CST V: L. jensenii). Microbiota dominated by Lactobacillus species are characterised by a low vaginal pH (<4.5), which is driven by lactic acid produced by these microorganisms.4 In contrast, CST IV is characterised by a paucity of Lactobacillus spp. and a diverse array of strict and facultative anaerobes such as Gardnerella vaginalis, Atopobium vaginae and Prevotella, among others.1 3 Interestingly, CST IV microbiota is frequent in women who are asymptomatic and otherwise healthy, but reminiscent of those in women diagnosed with bacterial vaginosis (BV). They are also characterised by relatively high pH.1 The Nugent score, a microscopic evaluation of bacterial morphotypes of a Gram stain vaginal smear, is routinely used to assess BV, referred to as Nugent-BV.5 Nugent-BV is associated with an increased risk for the acquisition of several STIs, including the human papillomavirus (HPV) and human immunodeficiency virus (HIV).6–8 Studies have shown that Nugent-BV is also associated with several population characteristics, such as ethnicity, sexual behaviour and certain contraceptive methods.9–11 Nugent-BV is closely associated with CST IV, and as expected, they share similar associations with women’s sociodemographic and behavioural characteristics.12–14

Interestingly, women with microscopically detected BV may also present with L. iners-dominated vaginal microbiota, often referred to as CST III.3 14 The association between Nugent-BV and CST III might be driven by the polymorphic nature of L. iners which can present as small Gram-negative coccobacilli due to its thinner peptidoglycan layer when compared with other Lactobacillus spp.15 Such microscopic features of L. iners are similar to G. vaginalis which contributes to Nugent score.5 The protective role of L. iners in the vaginal environment has been considered as suboptimal, as it offers limited protection against Chlamydia trachomatis, HPV and HIV.16–19 In addition, L. iners-dominated microbiota is associated with instability and frequently shifts to Lactobacillus-deprived CST IV and vice versa.2 16–20 Of the four most frequent vaginal Lactobacillus spp., L. iners is unique. It has a smaller genome size,21 does not produce hydrogen peroxide, nor D-lactic acid, producing only L-isomer of lactic acid,22 has cytotoxic capabilities through the production of inerolysin, a pore-forming cholesterol-dependent cytolysin.23 24

Considering the high prevalence rates reported for L. iners-dominated CST III in many populations worldwide, including Brazil,14 these observations are concerning. Thus, given the potential role for L. iners in vaginal microbiota instability and increase risk for STIs, we aimed to identify sociodemographic, behavioural and clinical characteristics in Brazilian women that are associated with L. iners-dominated CST III.


Study design and population

The study leveraged data from a subset of participants from a prior study that aimed to characterise the vaginal microbiota of Brazilian woman of reproductive age and to identify the characteristics associated with Lactobacillus-depleted microbiota, that is, CST IV.14 In the parent study, we cross-sectionally enrolled 609 participants from five geographical regions in Brazil (South, n=109; Southeast, n=140; Midwest, n=119; North, n=133; and Northeast, n=108), from 2012 to 2014.14 These women were approached when attending primary healthcare clinics, except for in the South region where enrolment was performed through a referral hospital. Only women presenting for routine cervical cancer screening (Pap testing) were considered for enrolment.

Participants were enrolled if they were between 18 and 50 years old, were not pregnant and did not report use of intrauterine device, immunosuppressive therapy or current urinary tract infection. Samples were collected at a minimum of 5 days after the end of last menstrual period, at least 72 hours since last sexual intercourse. Participants did not report use of antimicrobial drugs in the 45 days prior to enrolment.

Data collection and sampling procedures

Data and biological sample collection were as previously described in the parent study.14 Briefly, before the clinical examination, a face-to-face interview was administered using a structured questionnaire that comprised information regarding sociodemographic, behavioural characteristics and clinical history.14

After insertion of a non-lubricated speculum, two sterile swabs were rolled on the middle third of the vaginal wall. The first swab was stored in Amies liquid transport medium (Copan, Brescia, Italy) at −80°C for the characterisation of the vaginal microbiota composition. The other vaginal swab was smeared onto glass slides for Nugent scoring.5 The same swab was used for KOH testing, adding two drops of KOH solution with results reported as positive, inconclusive or negative by the nurse/physician depending on the presence of volatile amines (‘whiff test’). Assessment of vaginal pH was performed by applying a pH strip to the mid-vaginal wall for 1 min and comparing with the colour scale provided by the manufacturer (Merck, Darmstadt, Germany). Cervical brush samples were taken for C. trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis testing by PCR according to methods previously described.14

Nugent scoring and microbiota analysis

Vaginal smears were Gram-stained and further classified according to Nugent et al. (1991)5 The presence of presumptive Candida spp. morphotypes (pseudohyphae and/or hyphae) was recorded. The number of polymorphonuclear (PMN) inflammatory cells were counted in at least 20 fields for each smear, and results were expressed as average number of cells per field.

Total bacterial DNA was extracted from swabs stored in Amies transport medium using MoBio Powersoil Kit (MoBio Lab, Carlsbad, California, USA) according to previous published and validated procedure.25 26 Microbiota characterisation was performed through the Microbiome Service Laboratory at the Institute for Genome Sciences, University of Maryland School of Medicine in Baltimore, Maryland, using the methodology developed by Fadrosh et al. (2014)25 The V3-V4 regions of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq platform (Illumina). Sequence analysis details were previously reported by Marconi et al. (2020)14 Study samples were then clustered into CSTs based on their taxonomic composition, taxa relative abundances and Jensen-Shannon divergence metrics, according to methods previously described.1 3 As previously detailed,14 CST III was dominated by L. iners over other bacterial taxa, while the dominating taxa in CSTs I, II and V were, respectively, L. crispatus, L. gasseri and L. jensenii. For the analysis of this study, participants with L. iners CST III (n=222) were compared with those with non-L. iners dominated CSTs (CSTs I, II and V) (n=220).

Statistical analysis

Descriptive statistical analyses were performed for sociodemographic, behavioural and clinical variables between CST III and others Lactobacillus spp.-dominated CSTs using χ2 and Mann-Whitney tests for categorical and continuous variables, respectively, with a p value <0.05 considered as statistically significant.

We performed univariate logistic regression analysis to test the association between CST III and participant characteristics. Crude and age-adjusted and region-adjusted OR were estimated, as well as the corresponding 95% CIs. A multivariable logistic regression analysis was also performed using a forward stepwise model (variables were retained at p value ≤0.15) with CST III set as dependent variable. All analyses were performed in Stata/SE, V.15.1 (Stata Corp, College Station, Texas, USA).


Sociodemographic and behavioural data of the 442 women included in the two study groups are displayed in table 1. For both groups, median age of participants was 34 years old and slightly more than half of them self-reported as having a black or other alike skin colour. The proportion of women with CST III with completed high school was lower (52.70%), when compared with other CSTs (66.36%) (p=0.003). Regarding the behavioural characteristics, nearly all (94.55%) women with CSTs I/II/V reported intake of milk and dairy products. Condom use as contraceptive method was reported by less women with CST III (29.28%) when compared with other CSTs (40.00%) (p=0.018). Participants who reported having two or more sexual partners in the years prior to enrolment were frequent in CST III (11.26%) when compared with other CSTs (5.00%) (p=0.009). In relation to the clinical characteristics displayed in table 2, frequency of Nugent-BV (12.16%), increased number of inflammatory cells (10.81%) and presence of Candida morphotypes (pseudohyphae and/or hyphae) (9.91%) on vaginal smears were superior in CST III when compared with other vaginal microbiota types (p<0.05). The overall prevalence rates for C. trachomatis, N. gonorrhoeae and T. vaginalis were, respectively, 4.1% (n=18), 0.4% (n=2) and 0.2% (n=1). The prevalence of these infections was similar in the two study groups (table 2).

Table 1

Sociodemographic and behavioural variables grouped into CST I, II and V vs CST III

Table 2

Clinical variables grouped into CST I, II and V vs CST III

Crude logistic regression analysis (table 3) showed that CST III was over-represented among women enrolled in the northeast region (OR 1.98; 95% CI 1.09 to 3.60). Participants with CST III showed lower education level (OR 0.56; 95% CI 0.38 to 0.82), less consumption of milk and derivatives (OR 0.43; 95% CI 0.21 to 0.88) and lower rates of condom use (OR 0.62; 95% CI 0.41 to 0.92). Further, CST III was associated with having two or more sex partners in the past 12 months (OR 2.52; 95% CI 1.20 to 5.27). The age-adjusted and region-adjusted logistic regression analysis showed very similar associations than those obtained with the crude analysis, except that this model identified a positive association between CST III with report of prior episode of BV (OR 1.62; 95% CI 1.08 to 2.43).

Table 3

ORs and 95% CIs for the association between sociodemographic and behavioural factors Lactobacillus iners-dominated vaginal microbiota (community state type III)

Multivariable analysis (table 3) showed that CST III was independently associated with having two or more sex partners when compared with one sex partner (OR 3.27; 95% CI 1.50 to 7.11) and with the presence of Candida on vaginal smears (OR 2.24; 95% CI 1.02 to 4.89). Some sociodemographic and behavioural characteristics such as higher education level (OR 0.61; 95% CI 0.41 to 0.91), consumption of milk and derivatives (OR 0.43; 95% CI 0.20 to 0.90) and condom use (OR 0.59; 95% CI 0.38 to 0.91) were inversely associated with CST III vaginal microbiota.


Data from previous clinical and laboratory studies suggest that L. iners-dominated vaginal microbiota, often referred to as CST III, may not be optimal for women’s reproductive health.16–18 Understanding population characteristics associated with vaginal microbiota dominated by L. iners is thus critical for the management of adverse outcomes associated with this type of microbiota. In this study, we identified several characteristics associated with L. iners-dominated CST III in a cohort of Brazilian women. The proportion of women with CST III was lower among those who reported having a high school degree, consuming milk/dairy and using condom during sex, while CST III was significantly more frequent among participants with more than one sex partner in the year prior to enrolment. Interestingly, another factor associated with L. iners-dominated CST III was the presence of pathogenic forms of Candida spp. on vaginal smears by microscopic observation. However, we should consider that this is a cross-sectional study, and this study limitation does not allow to address any causal links between participants’ characteristics and type of vaginal microbiota.

The current findings demonstrated a novel association between a CST III microbiota and the lower scholar degree, that is, participants who did not complete high school. Education is one of the main socioeconomic indicators and is well acknowledged as predisposing to several STIs.27 Other studies have shown that STI acquisition is increased in the presence of vaginal microbiota associated with BV, which, combined with our results, indicates an association between socioeconomic characteristics and the composition of the microbiota.8–10 Because L. iners-dominated CST III have been associated with shifts to Lactobacillus-deprived CST IV types and vice versa,3 16 20 these two types of microbiota are not completely independent, and it is not surprising that we also identified lower education as a factor associated with CST III vaginal microbiota.

Similarly, sexual behaviour was shown to be significantly associated with CST III. This factor is also associated with BV including high number of sexual partners and inconsistent condom use.28 Current data showed the association between having two or more sex of partners in the previous year with CST III, in relation to one sex partner that was used as reference category due most of women included in this study referred having one sex partner. Although only 8.1% of the study participants reported having two or more sex partner, current observation agrees with previous study that reported increased prevalence of L. iners among African women with two or more sexual partners within 3 months prior to enrolment.12 Regarding the use of condom, our data are in agreement with findings by Vodstrcil et al. (2017) who showed that unprotected sex is associated with vaginal microbiota dominated by L. iners or BV-associated G. vaginalis.29 Interestingly, L. crispatus-dominated CST I was significantly associated with reporting consistent condom use.30 Despite the significant associations reported herewith, our understanding of the role of a woman’s sexual behaviour on the composition of the vaginal microbiota is not complete and should be explored further. A study limitation was that sexual behaviours were assessed through face-to-face interviews, and due to the sensitive nature of the questions, some participants may have omitted some information. This might explain the lack of association between CST III and other behaviours such as new sex partner and the frequency of sexual intercourse, which have been previously shown to be associated with CST IV.14

Studying diet and the vaginal microbiota is emerging, as diet is a modifiable factor that is thought to modulate the composition of the vaginal microbiota.31 32 Here we found an inverse association between milk and dairy intake and L. iners-dominated microbiota. Recently, our study group showed inverse association between milk and dairy intake with Lactobacillus-depleted CST IV.14 Other studies have shown that BV is associated with a low intake of vitamins and minerals often present in milk such as vitamin D, vitamin E and calcium,33 34 but no study has investigated these factors and the composition of the vaginal microbiota. It is well acknowledged that the composition of the gut microbiota is impacted by diets.35 Although a few studies point to an association between vaginal microbiota and host’s diet, this topic has not been as investigated as the relation between diet and gut microbiome. One of the few studies available was performed by Laue et al. (2018) and showed that the intake of a probiotics-containing yoghurt improved the remission rate and symptoms of BV.36 Another study using a mouse model showed that a Saccharomyces cerevisiae-based probiotic decreased vaginal loads of G. vaginalis.37 Thus, the impact of diet on vaginal microbiota suggests a potential association between its components with those colonising the gut. However, the underlying mechanisms of the association between vaginal and gut microbiota are poorly understood and should be investigated in detail by further studies. In fact, we are aware of this study limitation, as it fails to provide more detailed information regarding milk/dairy intake such as quantities and type of products more frequently consumed (yoghurt, cheese, fermented milk) by the study population.

Regarding the clinical characteristics, the association between microbiota dominated by L. iners and increased number of inflammatory cells on vaginal smears had not been reported previously. Thus, we have hypothesised that increased PMN cells in CST III could be due to a concurrent cervical infection. However, we have not observed any association between CST III and infections caused by C. trachomatis, N. gonorrhoeae and T. vaginalis infections in the present study. Recently, van Houdt et al. (2018) showed that women with L. iners-dominated microbiota have an increased susceptibility to C. trachomatis infection,18 which could be due to the incapability of D-lactic acid production by L. iners.19 However, we should consider L. iners produces the L-isomer of lactic acid, which may also play a protective role against infection.38 We recognise that the increased PMN cells on vaginal smears could also be due to the presence of other agents of cervical infection (eg, Mycoplasma genitalium) or other vaginal colonisers that were not addressed by this study. However, because of the low frequency of cervicitis in the population and the association shown between CST III and Candida spp. morphotypes on vaginal smears, we find it reasonable to attribute to the latter agents the most probable cause of the increased number of PMN cells in CST III.

In fact, a study conducted in three African countries demonstrated a higher prevalence of vaginal candidiasis, diagnosed by wet mount microscopy, in L. iners-dominated vaginal microbiota, although this finding was not statistically significant.13 Recently, Tortelli et al. (2019) showed that L. iners-dominated microbiota were more likely to test positive for Candida sp. using quantitative real-time PCR than L. crispatus-dominated community.39 On the other hand, a study by Brown et al. (2019) showed that CST dominated by L. iners had lower number of samples with C. albicans detected by PCR, while CST dominated by L. crispatus were most likely to present C. albicans.40 It is worth mentioning that PCR detects presence of Candida in minimum amounts, while microscopy is less sensitive. Thus, we hypothesised that L. crispatus could allow the colonisation by Candida sp. but might inhibit its growth and development. Alternatively, L. iners may not be as protective and could allow for the progression of Candida infection, thus explaining the presence of higher number of inflammatory PMN cells on vaginal smears of CST III. This hypothesis is supported by the study of Tortelli et al. (2019) that showed an increased in vitro inhibition of C. albicans growth by L. crispatus when compared with L. iners.39


L. iners-dominated CST III is associated with behavioural and socioeconomic characteristics (number of sexual partners, regular use of condoms and education level) that could be easily assessed in gynaecology clinics. Microscopic observation of Candida morphotypes on vaginal smears may also be helpful for identifying women with CST III. Considering that the protective role of L. iners has been questioned, the identification of women presenting this type of microbiota might be of clinical interest. However, there is no therapeutic option that target L. iners, and thus the information is not actionable currently. Further studies are necessary to identify novel strategies to modulate the vaginal microbiota for maintenance of a protective and beneficial environment to women’s reproductive health.

Key messages

  • Dominance of Lactobacillus iners in the vaginal microbiota is associated with low dairy intake and sex behaviour.

  • L. iners may offer a suboptimal protection against vaginal colonisation by Candida sp.

  • Easily available sociodemographic/behavioural characteristics may help in identifying women with a potentially more vulnerable state of the vaginal microbiota.

Abstract translation

This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

Data availability statement

No data are available. Not available.

Ethics statements

Patient consent for publication

Ethics approval

The study was reviewed and approved by the Ethics Committee of Botucatu Medical School (approval numbers: 3.094.514 and 294.202). After being informed of the aims and procedures of the study, all participants signed a written informed consent.


Supplementary materials

  • Supplementary Data

    This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.


  • Handling editor Francesca Ceccherini-Silberstein

  • Contributors CM and MGS designed the study. CSTF, ART and CM lead the clinical study and biological sample collection. JN, CM and CSTF performed laboratory analysis. JR and BM obtained and processed sequencing data. JN and CM conducted the statistical analysis and interpreted the data. JN drafted the manuscript under the supervision of CM and MGS. JR and AdRT edited the manuscript. All authors reviewed and approved the submitted version of the manuscript.

  • Funding This study has received funding by developmental funds granted by São Paulo Research Foundation − FAPESP (grant number 2012/16800-3) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior − Brasil − CAPES (Master’s scholarship number 1680048).

  • Disclaimer The sponsor not been involved in the conduct of the research or the preparation of the article.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.