The Presence of Lactobacillus spp. and its Effect on the Occurrence of Other Microorganisms in the Reproductive Tract of Polish Women

The vaginal microbiota of healthy women of reproductive age is characterized by low biodiversity and a dominance of species from the genus Lactobacillus (Cadieux et al. 2002; Ravel et al. 2011; Kasprowicz and Białecka 2016). The most common species of lactic acid bacilli inhabiting the vagina include Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus iners (Witkin et al. 2021). Due to the prevalence of individual lactobacilli species, Ravel et al. (2011) developed the community state types (CST) classification, which distinguishes several types of vaginal microbiota. CST I, CST II, CST III, and CST V are dominated successively by L. crispatus, L. gasseri, L. iners, and L. jensenii. CST IV represents a group in which Lactobacillus spp. is not the dominant species. There is a distinction between CST IV-A characterized by the presence of a small number of L. iners and CST IV-B with a predominance of bacteria such as Fannyhessea, Gardnerella, Mobiluncus, Prevotella, and Sneathia (Ravel et al. 2011). France et al. (2020) used an extensive database of approximately 13,160 vaginal microbiota profiles obtained by the 16S rRNA gene amplicon sequencing. Researchers have divided vaginal microbiota profiles into CST I, II, III, and V which are dominated successively by L. crispatus, L. gasseri, L. iners, and L. jensenii. CST I and CST III were then divided into A and B, version A had a higher number of bacteria, while version B had a lower number of bacteria. CST-IV was divided into CST IV-A, IV-B, and IV-C dominated by “Candidatus Lachnocurva vaginae”, Gardnerella vaginalis, a group with a predominance of facultative and strict anaerobic species. CST-IV was further divided into five subtypes: CST IV-C0, CST IV-C1, CST IV-C2, CST IV-C3, and CST IV-C4 which are dominated successively by Prevotella, Streptococcus, Enterococcus, Bifidobacterium, and Staphylococcus. Then, collected reference data was used to train a nearest centroid classifier VALENCIA (VAginaL community state typE Nearest CentroId clAssifier) to appropriately classify the vaginal microbiota profile. The VALENCIA closest center of gravity classification algorithm was created, allowing for the repeatable and accurate assignment of vaginal microbiota to appropriate CSTs. VALENCIA is a tool that uses the closest centroid for analysis, classifying samples based on similarity to a set of reference centroids. VALENCIA provides reliable and reproducible CST assignment by constructing a reference centroid and nearest centroid classification algorithm (France et al. 2020).

Lactic acid bacilli in the vagina perform protective functions through the production of lactic acid, hydrogen peroxide (H₂O₂), and bacteriocins (Gajer et al. 2012; Tachedjian et al. 2018; Zheng et al. 2021). Lactic acid is found in two varieties L-lactic acid and D-lactic acid, of which D-lactic acid has a stronger protective effect. L-lactic acid blocks histone deacetylase, stimulating gene transcription and DNA repair. H₂O₂ in vitro shows high activity against Staphylococcus aureus, Escherichia coli, Candida albicans. Lack of H₂O₂ in the vagina is hypothesized to increase the number of catalase-negative bacteria, which may contribute to the risk of bacterial vaginosis and human immunodeficiency virus (HIV) infection (Knezević et al. 2005). Bacteriocins, on the other hand, kill pathogens by blocking DNA synthesis. Not every Lactobacillus species provides a protective barrier. It concerns, for example, the species of L. iners (Aroutcheva et al. 2001; Vallor et al. 2001; Amabebe and Anumba 2018). Witkin et al. (2021) showed that the presence of L. iners was associated with more frequent colonization by pathogenic bacteria (Witkin et al. 2021) and does not protect against infection by Chlamydia trachomatis (Edwards et al. 2019). Lactobacilli, due to their protective properties, inhibit the proliferation of pathogenic microorganisms, counteracting the occurrence of bacterial vaginosis, among others. Bacterial vaginosis can contribute to serious health problems, including an increased risk of infertility, C. trachomatis infection, gonorrhea, human papilloma virus (HPV) and HIV, as well as pelvic organ inflammation (PID) and fallopian tube inflammation (Ravel et al. 2021). In addition, studies show that there is a relationship between decreased number of lactobacilli and the incidence of preterm labor, miscarriage and postpartum bleeding, and reduced fertility (Newton et al. 2001; Ness et al. 2004; Galhardo et al. 2006; Witkin and Linhares 2017; Kiecka et al. 2021). Hence, information on the incidence of specific etiological agents of genital tract infections is valuable from a clinical perspective (Mączyńska 2008).

Women of reproductive age were selected as the study group. The aim of this study was to determine whether the number of Lactobacillus spp. can contribute to the inhibition of reproductive tract infections among women who report to the microbiology laboratory for check-ups or report due to emerging symptoms. Additionally, the aim was to determine how the presence of Lactobacillus spp. affects the occurrence of symptoms and the pH value.

This retrospective study presents the results of women of reproductive age (18–49 years old) diagnosed at the Centre for Microbiology and Autovaccines in Cracow, Poland. The relationship between vaginal colonization by specific microorganisms and the occurrence of clinical symptoms in the form of burning, itching, discomfort, discharge and pain was investigated. The number of Lactobacillus spp. increases, and the risk of symptoms decrease. It confirms the protective role of Lactobacillus spp. (Boskey et al. 2001; Ravel et al. 2011).

The occurrence of candidiasis was observed to influence the appearance of symptoms. According to Mendling et al. (2015) candidiasis is associated with itching, discharge, burning sensation, and redness in only half of the cases. Often, women have asymptomatic Candida colonization, which requires treatment only before delivery to avoid obstetric complications (Mendling et al. 2015). In addition, women with symptomatic reproductive tract infections with C. trachomatis were found to be diagnosed more than 13 times more frequently. This may be related to the fact that C. trachomatis infection is associated with pelvic organ inflammation (Gottlieb et al. 2013). Additionally, colonization by U. parvum, M. hominis, and U. urealyticum was associated with symptoms in women of reproductive age. Our previous study showed that ureaplasmas are closely associated with the onset of symptoms among women (Rak et al. 2022). However, symptoms do not always indicate infection with pathogenic microorganisms (Mitchell et al. 2020). This can also be seen in this data analysis. Among the 1,039 women studied, 88 were symptomatic, and there was no colonization by pathogenic microorganisms. Among these 88 symptomatic women, 10 had Lactobacillus spp. on 0–1, which, in theory, could explain the appearance of itching, burning, and pain. However, up to 78 symptomatic women had Lactobacillus score of 2 or 3 in the vagina, which is a good result, and the vaginal pH was normal. However, women reported discomfort, pain, and burning. It may be due to other factors that may influence the symptoms, including hormonal fluctuations, allergies, environmental factors, phase of the menstrual cycle, and sexual intercourse.

It is well known that lactic acid bacteria can adhere and compete for adhesion sites in the vaginal epithelium. In addition, they produce several antimicrobial compounds (lactic acid, bacteriocins, and hydrogen peroxide) that inhibit vaginal colonization by pathogenic microorganisms (Mei and Li 2022). Similar to Schwebke et al. (2014), it was shown that in women, the number of Lactobacillus spp. increases, colonization by G. vaginalis is less frequent and more lactic acid bacilli, the less frequent the infection by Streptococcus group B and Enterobacterales. However, this relationship was not observed for ureaplasm and mycoplasm.

According to Amsel’s criteria (Amsel et al. 1983), pH is one of the predictive factors of bacterial vaginosis. A relationship between Ureaplasma spp. and elevated vaginal pH was demonstrated in our previous study (Rak et al. 2022). This retrospective analysis also showed a correlation between increased vaginal pH and colonization by U. urealyticum, G. vaginals, Enterobacterales, and Streptococcus group B. In addition, a trend was identified: with an increase in Lactobacillus, there is a decrease in pH.

The strengths of this retrospective analysis were the large number of patients and the knowledge of microbiological composition of the reproductive organs of each patient. This study showed that even the use of classical diagnostic microbiology can expand our knowledge of the prevalence of microorganisms in the genital tract and affect the health of women of reproductive age.

Limitations of this study include the fact that the species of Lactobacillus spp. found in the vagina have not been identified. Since their identification could shed new light on the relationship between lactobacilli and vaginal health. Future research should focus on determining which species of Lactobacillus spp. provide a protective function. For example, in recent years, it has been increasingly reported that L. iners, among others, may represent a transitional species between a healthy vagina and a tendency to vaginal infections (Kumar et al. 2023). A limitation of this study is that complete information about the patients is not known, such as sexual behavior, complete medical history, and socio-economic characteristics. An important fact is that in our study, asymptomatic women with Lactobacillus spp. defined out of 3 sometimes have a vagina colonized by pathogenic organisms. It shows how important it is to perform preventive examinations. Undiagnosed infections of the reproductive organs can cause obstetric complications in the form of miscarriages, premature births, and ectopic pregnancies. Farr et al. (2015) showed that a lack of Lactobacillus spp. in the vagina and colonization with pathogenic bacteria increases the risk of preterm delivery and low birth weight in women in early pregnancy.

In conclusion, the relationship between Lactobacillus spp. and the inhibition of colonization of reproductive organs by pathogenic microorganisms was found. It was shown that lactobacilli significantly reduce the risk of symptoms among women. However, further analysis of co-infections and associations between symptom occurrence in female reproductive tract infections is needed.