
==== Front
Pol J Microbiol
Pol J Microbiol
pjm
pjm
Polish Journal of Microbiology
1733-1331
2544-4646
Sciendo

39213132
pjm-2024-024
10.33073/pjm-2024-024
Original Paper
The Presence of Lactobacillus spp. and its Effect on the Occurrence of Other Microorganisms in the Reproductive Tract of Polish Women
https://orcid.org/0000-0002-3818-8972
Kiecka Aneta 12aneta.kiecka@uj.edu.pl

https://orcid.org/0000-0003-4634-619X
Rak Katarzyna 2
https://orcid.org/0000-0002-0820-3174
Białecka Joanna 23
https://orcid.org/0000-0003-2772-6787
Białecka Anna 2
https://orcid.org/0000-0002-0388-2382
Szczepanik Marian 1
1 Jagiellonian University Medical College, Faculty of Health Sciences, Institute of Physiotherapy, Chair of Biomedical Sciences, Cracow, Poland
2 Centre of Microbiological Research, Cracow, Poland
3 Jagiellonian University Medical College, Department of Pharmaceutical Microbiology, Cracow, Poland
25 8 2024
9 2024
73 3 265273
5 2 2024
24 5 2024
© 2024 Aneta Kiecka et al., published by Sciendo
2024
Aneta Kiecka et al., published by Sciendo
https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Abstract

Disorders of the vaginal microbiota can lead to many complications and affect fertility. This study evaluates the role of Lactobacillus in the vagina and its impact on the incidence of colonization by pathogenic microorganisms, analyzing the results of 1,039 women of reproductive age (18–49 years) who underwent a microbiological examination of the reproductive tract in 2020. Samples were examined by microscopy, culture, and NAAT. As the number of Lactobacillus increases, the chance of developing symptoms decreases. In fact, it has been shown that the higher the number of Lactobacillus spp. the less frequently Gardnerella vaginalis and Streptococcus group B are observed. As the concentration of Lactobacillus spp. increases by one category, the risk of G. vaginalis after adjustment to age and pH decreases by 80% (p < 0.001). Similarly, the correlation between Lactobacillus spp. and vaginal pH was shown. After adjustment to age, the odds of prevalence pH > 4.5 for people with Lactobacillus category higher 1 is 76% lower.

Keywords

female reproductive tract microbiota
Lactobacillus
bacterial vaginosis
genital infections
==== Body
pmcIntroduction

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.

Experimental

Materials and Methods

Study population

The study was a retrospective analysis of the results of 1,039 microbiological tests of the genital tract among women of reproductive age from 18 to 49 years of age at the Center of Microbiology and Autovaccines in Cracow, Poland, from April to August 2020. The study was carried out with the consent of the Bioethics Committee No. 118.6120.32.2023. The analysis included women who underwent a complete package of tests, including vaginal culture, evaluation of microscopic preparation of vaginal biocenosis, evaluation of pH value of vaginal contents, testing for C. trachomatis, Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium. In addition, a detailed medical history was recorded (all symptoms and treatment reported by the patients, including antibiotic therapy, were noted), and these data were obtained from the patient’s medical records and attending physicians. The data used for the analysis were age, symptoms/absence, pH of vaginal contents, microbiological culture results, and molecular testing results (NAAT). Exclusion criteria were as follows: age criteria not met, under 18 and over 49 years of age, pregnant women, and antibiotic therapy in the last 2 months.

Collection of test material

Samples were taken from vagina and the cervical canal. Test material consisted of two vaginal swabs (one for microscopic preparation, pH and culture and the other for Trichomonas vaginalis culture) and one cervical swab for NAAT.

Determination of the pH value of vaginal contents

During the collection of vaginal swabs, 0.1 g of vaginal content found in the vaginal speculum was taken to determine the pH value of vaginal content. The pH of vaginal contents was measured using indicator strips in the range of 2.0–9.0 pH (Merck KGaA, Germany). The pH was then recorded in the patient’s record. Normal vaginal pH ranges from 3.5 to 4.5. A pH above 4.5 indicates an abnormal picture of vaginal biocenosis.

Evaluation of microscopic preparation

One of the swabs with vaginal contents was used for preparation. The collected vaginal contents were carefully spread on a microscope slide. The slide was then stained by the Gram method using a PREVI® Color Gram apparatus (bioMerieux, France). Microscopic preparations were evaluated using the Kuczyński scale (Kuczyńska 2003) with Kasprowicz modification (Kasprowicz and Białecka 2016). The presence of Lactobacillus spp. was evaluated on a scale of 0–3, where 0 means none, 1 –a single bacterium (up to 10 in the field of view), 2 – few (11–50 in the field of view), and 3 – many bacteria (> 50 in the field of view) (Fig. 1). The presence of leukocytes, bacteria, and fungi on a scale of 0–1 was also determined.

Fig. 1. Microscope photos showing Lactobacillus categories of 0, 1, 2, and 3.

A) 0 – means none, B) 1 – a single bacterium (up to 10 in the field of view), C) 2 – few (11–50 in the field of view), D) 3 – many (> 50 in the field of view) cells of Lactobacillus.

Detecting the presence of T. vaginalis

The swab was examined in 2 ml of Trichomedium (GRASO® Biotech, Poland). 48 hours later, a direct preparation was made. The preparation was observed under a microscope for identification of T. vaginalis.

Culture

A swab of vaginal contents was used to culture by direct plating the Petri dish on TSA media with 5% sheep blood (GRASO® Biotech, Poland), Gardnerella Agar (bioMerieux, France), CANDISelect (Bio-Rad, USA), and Chocolate Agar + P.V.S. + VCAT (Bio-Rad, USA). Then, the media was incubated for 24 hours or 48 hours at 37°C. After this time, microorganisms were identified using MALDI-TOF (Bruker Daltonics GmbH & Co., Germany) or VITEK® 2 (BioMerieux, France).

NAAT

A cervical contents swab was placed in 3 ml of UTM® medium (Copan Italia s.p.a., Italy). DNA was isolated using croBEE 201A (GeneProof a.s., Czech Republic) and croBEE NA16 (GeneProof a.s., Czech Republic) for nucleic acid extraction. NAAT assay was conducted using AmpliSens PCR assay in a Cobas® Z480 real-time thermocycler (Roche, Switzerland). NAAT was used to detect C. trachomatis, U. parvum, U. urealyticum, M. hominis, and M. genitalium.

Statistical analysis

An R language, version 4.0.4 (R Core Team 2021), an environment for statistical computing, was used for all statistical analysis. The two-way p-value = 0.05 was set as a level of significance. The pH distribution was characterized by the median and interquartile range and was compared between women with or without the prevalence of particular microorganisms using the Mann-Whitney U test. The occurrence of microorganisms, symptoms, or pH > 5 was expressed in percentages and counts and compared across independent groups with the Fisher exact test. Additionally, univariable and multivariable logistic regression was applied for dichotomous outcomes (such as symptoms, pH > 4.5, prevalence of microorganisms). Due to the frequent occurrence of small cell counts in contingency tables showing the joint distribution of microorganisms and particular outcomes, a penalized likelihood Firth’s method was applied to calculate odds ratios with 95% confidence intervals (ORs and CIs). p-Values were based on the Wald test. Variables used for adjustment were based on the literature and were limited only to the most important confounders.

Results

Study patients’ population

The study group consisted of 1,039 women aged 18–49, among whom 287 showed burning, itching, pain, discomfort, and discharge symptoms, while 752 patients showed none. Lactobacillus spp. (borderline 1–3) was found in 993 women, of which 132 women had Lactobacillus spp. with a score of 1, 611 women with a score of 2, and 250 women – a score of 3 (for reference here, see paragraph Evaluation of microscopic preparation) Lactobacillus spp. was absent in 46 patients. G. vaginalis was found in 51 women in the study group. Enterobacterales was detected in 55 women, Streptococcus group B in 81, and yeast-like fungi in 177 patients. The presence of Ureaplasma spp. was found in 245 women, of which U. parvum was identified in 165 cases, while U. urealyticum in 80 cases. M. hominis was reported in 19 women and M. genitalium in 1, while C. trachomatis was reported in eight women. This study did not show Neisseria gonorrhoeae and T. vaginalis in in any patient. Complete data on infections present in women are shown in Table I.

Table I Occurrence of individual microorganisms in women with and without symptoms qualified for the study and the microbiological methods used.

Microorganisms	Method	Total	Without symptoms (n = 752)	With symptoms (n = 287)	p-value*	OR# (95% CI)	p-value**	
n (%)	n (%)	n (%)	
Lactobacillus spp.†	0	microscopic	46 (4.4%)	19 (2.5%)	27 (9.4%)	< 0.001	1 (REF.)	–	
1	132 (12.7%)	90 (12.0%)	42 (14.6%)	0.33 (0.17; 0.66)	0.002	
2	611 (58.8%)	451 (60.0%)	160 (55.7%)	0.25 (0.14; 0.46)	< 0.001	
3	250 (24.1%)	192 (25.5%)	58 (20.2%)	0.22 (0.11; 0.41)	< 0.001	
Gardnerella vaginalis	culture, microscopic	51 (4.9%)	11 (1.5%)	40 (13.9%)	< 0.001	10.55 (5.40; 20.63)	< 0.001	
Ureaplasma parvum	PCR	165 (15.9%)	81 (10.8%)	84 (29.3%)	< 0.001	3.42 (2.43; 4.82)	< 0.001	
Ureaplasma urealyticum	PCR	80 (7.7%)	27 (3.6%)	53 (18.5%)	< 0.001	6.02 (3.71; 9.75)	< 0.001	
Mycoplasma hominis	PCR	19 (1.8%)	0 (0%)	19 (6.6%)	< 0.001	109.30 (6.58; 1,816.53)	0.001	
Mycoplasma genitalium	PCR	1 (0.1%)	0 (0%)	1 (0.3%)	0.276	7.88 (0.32; 193.98)	0.207	
Chlamydia trachomatis	PCR	8 (0.8%)	1 (0.1%)	7 (2.4%)	< 0.001	13.40 (2.31; 77.74)	0.004	
Enterobacterales	culture	55 (5.3%)	35 (4.7%)	20 (7.0%)	0.162	1.55 (0.88; 2.72)	0.127	
Streptococcus group B	culture	81 (7.8%)	57 (7.6%)	24 (8.4%)	0.698	1.12 (0.69; 1.84)	0.641	
Yeast	culture, microscopic	177 (17.0%)	100 (13.3%)	77 (26.8%)	< 0.001	2.39 (1.71; 3.34)	< 0.001	
Neisseria gonorrhoeae	culture	0 (0.0%)	0 (0.0%)	0 (0.0%)	–	–	–	
Trichomonas vaginalis	culture	0 (0.0%)	0 (0.0%)	0 (0.0%)	–	–	–	
* – all p-values are examined on Fisher’s test

** – p-values measured by Wald method using Firth’s logistic regression

# – OR of occurrence symptoms in women with or without detected microorganisms, reference category in all microorganism is “undetected”

† – 0 means none, 1 single (up to 10 in the field of view), 2 few (11–50 in the field of view), 3 many (> 50 in the field of view) cells of Lactobacillus

Microorganisms and clinical symptoms

Our data show that symptoms in the form of itching, burning, discharge, pain, and discomfort are associated with the presence of various microorganisms in the genital tract of women. Tables I and II show univariable and multivariable logistic regression models. In the crude model, women who had more Lactobacillus spp. were less likely to have symptoms. Specifically, the chance of clinical symptoms in women with Lactobacillus spp. scores equal to 1, 2 and 3 decreased by 67%, 75%, and 78%, respectively, compared to women with no Lactobacillus spp. Furthermore, it has been shown that when G. vaginalis colonize the vagina, there is a 10-fold increased risk of symptoms (OR = 10.55, 95% CI: 5.40; 20.63). All women in whom M. hominis was identified were symptomatic (OR = 109.30 6 95% CI: 6.58; 1,816.53). There was no significant relationship between the presence of M. genitalium and the onset of symptoms (p = 0.207), and the bacterium was diagnosed in only one patient. The results showed a positive correlation between the prevalence of fungi and symptoms (OR = 2.39, 95% CI: 1.71; 3.34). The prevalence of Streptococcus group B was not statistically different (p = 0.698) in symptomatic versus asymptomatic women, which was also confirmed by logistic regression (OR = 1.12, 95% CI: 0.69; 1.84). Multivariable analysis showed that the vaginal colonization by G. vaginalis, fungi, Mycoplasma spp., and C. trachomatis is corelated with symptoms. These results showed that Lactobacillus spp. have a protective role. After adjustment to age, pH and leucocytes the odds of clinical symptoms for women with Lactobacillus spp. score = 1 is lower by 67% compared to women with Lactobacillus spp. with a score = 0 (OR = 0.33, 95% CI: 0.16; 0.69, p = 0.002).The result for women with a higher number of Lactobacillus was at a similar level, with the trend of even stronger protective effect (the odds decreased by 73% and 77%, respectively).

Table II Relationship between the occurrence microorganisms and symptoms.

Microorganisms	Model 1	Model 2	Model 3	
OR# (95% CI)	p-value**	OR# (95% CI)	p-value**	OR# (95% CI)	p-value**	
Lactobacillus spp.†	1	0.31 (0.15; 0.61)	< 0.001	0.42 (0.20; 0.87)	0.019	0.33 (0.16; 0.69)	0.003	
2	0.23 (0.12; 0.43)	< 0.001	0.38 (0.19; 0.76)	0.006	0.27 (0.13; 0.54)	< 0.001	
3	0.20 (0.10; 0.38)	< 0.001	0.38 (0.18; 0.81)	0.012	0.23 (0.10; 0.50)	< 0.001	
Gardnerella vaginalis	10.47 (5.36; 20.47)	< 0.001	7.83 (3.93; 15.61)	< 0.001	10.03 (4.92; 20.47)	< 0.001	
Ureaplasma parvum	3.40 (2.42; 4.79)	< 0.001	3.18 (2.25; 4.50)	< 0.001	2.93 (2.06; 4.16)	< 0.001	
Ureaplasma urealyticum	5.98 (3.69; 9.69)	< 0.001	5.69 (3.49; 9.28)	< 0.001	5.56 (3.40; 9.09)	< 0.001	
Mycoplasma hominis	111.18 (6.71; 1,842.58)	0.001	98.35 (5.93; 1,631.18)	0.001	94.56 (5.72; 1,563.34)	0.001	
Chlamydia trachomatis	13.21 (2.28; 76.60)	0.004	13.56 (2.31; 79.70)	0.004	10.12 (1.69; 60.80)	0.011	
Yeast	2.38 (1.70; 3.32)	< 0.001	2.24 (1.59; 3.14)	< 0.001	1.93 (1.36; 2.74)	< 0.001	
1 Model 1 adjusted to age, Model 2 adjusted to age and pH, Model 3 adjusted to age, pH and leucocytes

# – OR of occurrence symptoms in women with microorganisms (except of Lactobacillus) compared with those without microorganisms, in the case Lactobacillus OR refers to prevalence of symptoms in women with detected

† – Lactobacillus at the level 1 single (up to 10 in the field of view), 2 few (11–50 in the field of view), 3 many (> 50 in the field of view) respectively, compared with 0 (lack)

** – p-values testing by Wald method and Firth’s logistic regression

Lactobacillus spp. and other microorganisms

Distribution of the prevalence of each microorganism according to the number of Lactobacillus spp. (0, 1, 2, 3). Table III shows statistically significant differences for G. vaginalis, Enterobacterales, and Streptococcus group B, with a decreasing prevalence trend among women with higher levels of Lactobacillus spp. In a multivariate analysis, it was confirmed that lactobacilli provide a protective barrier against colonization of the vagina by pathogenic microorganisms. Our study showed a correlation between the number of Lactobacillus spp. expressed as an ordinal variable (0, 1, 2, 3) and the incidence of G. vaginalis. As the concentration of Lactobacillus spp. increases by one category (on a scale of 0–3 – as defined in paragraph Evaluation of microscopic preparation), the risk of G. vaginalis, after adjustment to age and pH decreases by 80% (p < 0.001). Similarly, as the number of Lactobacillus spp. increases by 1 category, the chance of vaginal colonization by Enterobacterales decreases by 46% and by 37% by Streptococcus group B respectively after adjustment to age and pH (Table IV). When we considered Lactobacillus spp. as a dichotomous variable, without taking into account its number, a statistically significant result was observed only for G. vaginalis with a 79% reduction of odds prevalent of GV for women with the presence of Lactobacillus (at any number) compared to with those without Lactobacillus detected (Table IV).

Table III Relationship between the presence of Lactobacillus spp. and other microorganisms.

Microorganisms	Presence	n (%)	Lactobacillus spp.†	p-value*	
0	1	2	3	
Gardnerella vaginalis	No	988 (95.1%)	30 (65.2%)	108 (81.8%)	600 (98.2%)	250 (100%)	< 0.001	
Yes	51 (4.9%)	16 (34.8%)	24 (18.2%)	11 (1.8%)	0 (0%)	–	
Ureaplasma parvum	No	874 (84.1%)	36 (78.3%)	109 (82.6%)	520 (85.1%)	209 (83.6%)	0.542	
Yes	165 (15.9%)	10 (21.7%)	23 (17.4%)	91 (14.9%)	41 (16.4%)	–	
Ureaplasma urealyticum	No	959 (92.3%)	40 (87.0%)	119 (90.2%)	566 (92.6%)	234 (93.6%)	0.299	
Yes	80 (7.7%)	6 (13.0%)	13 (9.8%)	45 (7.4%)	16 (6.4%)	–	
Mycoplasma hominis	No	1020 (98.2%)	42 (91.3%)	129 (97.7%)	601 (98.4%)	248 (99.2%)	0.0153	
Yes	19 (1.8%)	4 (8.7%)	3 (2.3%)	10 (1.6%)	2 (0.8%)	–	
Mycoplasma genitalium	No	1038 (99.9%)	45 (97.8%)	132 (100%)	611 (100%)	250 (100%)	0.0443	
Yes	1 (0.1%)	1 (2.2%)	0 (0%)	0 (0%)	0 (0%)	–	
Chlamydia trachomatis	No	1031 (99.2%)	46 (100%)	131 (99.2%)	606 (99.2%)	248 (99.2%)	1.000	
Yes	8 (0.8%)	0 (0%)	1 (0.8%)	5 (0.8%)	2 (0.8%)	–	
Enterobacterales	No	984 (94.7%)	36 (78.3%)	112 (84.8%)	590 (96.6%)	246 (98.4%)	< 0.001	
Yes	55 (5.3%)	10 (21.7%)	20 (15.2%)	21 (3.4%)	4 (1.6%)	–	
Streptococcus group B	No	958 (92.2%)	34 (73.9%)	108 (81.8%)	571 (93.5%)	245 (98.0%)	< 0.001	
Yes	81 (7.8%)	12 (26.1%)	24 (18.2%)	40 (6.5%)	5 (2.0%)	–	
Yeast	No	862 (83.0%)	39 (84.8%)	111 (84.1%)	506 (82.8%)	206 (82.4%)	0.976	
Yes	177 (17.0%)	7 (15.2%)	21 (15.9%)	105 (17.2%)	44 (17.6%)	–	
Neisseria gonorhoeae	No	1039 (100%)	46 (100%)	132 (100%)	611 (100%)	250 (100%)	–	
Yes	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	–	
Trichomonas vaginalis	No	1039 (100%)	46 (100%)	132 (100%)	611 (100%)	250 (100%)	–	
Yes	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	–	
* – all p-values are testing on Fisher’s exact test

† – 0 means none, 1 single (up to 10 in the field of view), 2 few (11–50 in the field of view), 3 many (> 50 in the field of view)

Table IV The association between presence of Lactobacillus and other microorganisms – multivariable logistic regression models.

Microorganisms	Lactobacillus as ordered variable†	Lactobacillus as dichotomous variable††	
Per 1 category level increase	Not present	Present	Not present	Present	
Model 1	Model 2	Model 1	Model 2	
Yeast	1.04 (0.83; 1.30)	1.32 (1.02; 1.71)	1 (REF.)	0.97 (0.43; 2.18)	1 (REF.)	1.69 (0.70; 4.07)	
Enterobacterales	0.35 (0.25; 0.48)	0.54 (0.37; 0.81)	1 (REF.)	0.19 (0.09; 0.40)	1 (REF.)	0.78 (0.31; 1.97)	
Streptococcus group B	0.40 (0.30; 0.52)	0.63 (0.45; 0.89)	1 (REF.)	0.23 (0.11; 0.47)	1 (REF.)	0.99 (0.42; 2.29)	
Gardnerella_vaginalis	0.14 (0.09; 0.21)	0.20 (0.12; 0.32)	1 (REF.)	0.05 (0.02; 0.11)	1 (REF.)	0.21 (0.09; 0.51)	
Chlamydia_trachomatis	1.12 (0.45; 2.79)	1.39 (0.52; 3.75)	1 (REF.)	0.67 (0.04; 11.0)	1 (REF.)	1.19 (0.06; 24.33)	
Mycoplasma_hominis	0.47 (0.28; 0.78)	0.61 (0.32; 1.16)	1 (REF.)	0.16 (0.05; 0.49)	1 (REF.)	0.32 (0.08; 1.32)	
Ureaplasma_parvum	0.91 (0.72; 1.13)	1.16 (0.89; 1.50)	1 (REF.)	0.58 (0.28; 1.19)	1 (REF.)	1.02 (0.46; 2.26)	
Ureaplasma_urealyticum	0.75 (0.56; 1.01)	0.88 (0.62; 1.25)	1 (REF.)	0.45 (0.19; 1.07)	1 (REF.)	0.70 (0.26; 1.90)	
* – model 1 adjusted to age

** – model 2 adjusted to age and pH

† – Lactobacillus was coded as 0, 1, 2, 3, where 0 means none, 1 single (up to 10 in the field of view), few (11–50 in the field of view), 3 many (> 50 in the field of view) cells of Lactobacillus

†† – result for presence of Lactobacillus (1 + 2 + 3) compared to 0

Lactobacillus spp. and pH value

Out of 1,039 vaginal swabs, in 791 (76.1%) cases the pH was normal and in the range of 3.5–4.5. In 248 (23.9%) samples, the pH was above 4.5. Statistical analysis showed a strong relationship between the prevalence of high vaginal pH and the presence of G. vaginalis, U. urealyticum, Enterobacterales and Streptococcus group B, and yeast-like fungi (Table V). After adjustment to age, odds of prevalence pH > 4.5 for people with Lactobacillus category higher 1 is 76% lower. Distribution of pH between Lactobacillus categories was presented in Fig. 2.

Fig. 2. Distribution of pH between Lactobacillus categories.

Table V The pH values in women with and without the occurrence of microorganisms.

Microorganisms	Not prevalent	Prevalent	p *	pH > 4.5	p **	
n	Q2 (Q1–Q3)	n	Q2 (Q1–Q3)	OR (95% CI)**	
Lactobacillus spp.	46	5.5 (5.0; 5.5)	993	4.5 (4.0; 4.5)	< 0.001	0.01 (0.00; 0.05)	< 0.001	
Gardnerella vaginalis	988	4.5 (4.0; 4.5)	51	5.0 (5.0; 5.5)	< 0.001	12.18 (6.31; 23.48)	< 0.001	
Ureaplasma parvum	874	4.5 (4.0; 4.5)	165	4.5 (4.5; 5.0)	< 0.001	1.74 (1.21; 2.50)	0.003	
Ureaplasma urealyticum	959	4.5 (4.0; 4.5)	80	4.5 (4.4; 5.0)	0.009	2.36 (1.47; 3.78)	< 0.001	
Mycoplasma hominis	1020	4.5 (4.0; 4.5)	19	4.5 (4.5; 5.0)	0.014	2.88 (1.18; 7.01)	0.020	
Mycoplasma genitalium	1038	4.5 (4.0; 4.5)	1	5.0 (5.0; 5.0)	0.203	11.50 (0.46; 284.64)	0.136	
Chlamydia trachomatis	1031	4.5 (4.0; 4.5)	8	4.5 (4.4; 4.6)	0.698	1.27 (0.29; 5.52)	0.748	
Enterobacterales	984	4.5 (4.0; 4.5)	55	5.0 (4.5; 5.0)	< 0.001	6.10 (3.47; 10.74)	< 0.001	
Streptococcus group B	958	4.5 (4.0; 4.5)	81	5.0 (4.5; 5.0)	< 0.001	5.06 (3.17; 8.06)	< 0.001	
Yeast	862	4.5 (4.0; 4.5)	177	4.5 (4.5; 5.0)	0.001	1.66 (1.16; 2.37)	0.005	
* – based on Mann-Whitney U test

** – based on logistic regression, Firth’s method, crude models, OR of prevalence pH > 4.5 in women specific microorganisms compared with those without them; Q2 (Q1–Q3) denotes median (interquartile range)

Discussion

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.

Conflict of interest

The authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication.
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Literature

Amabebe E Anumba DOC. The vaginal microenvironment: The physiologic role of Lactobacilli . Front Med . 2018 NaN ; 5 : 181 . 10.3389/fmed.2018.00181
Amsel R Totten PA Spiegel CA Chen KC Eschenbach D Holmes KK. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations . Am J Med . 1983 NaN ; 74 ( 1 ): 14 – 22 . 10.1016/0002-9343(83)91112-9 6600371
Aroutcheva A Gariti D Simon M Shott S Faro J Simoes JA Gurguis A Faro S. Defense factors of vaginal lactobacilli . Am J Obstet Gynecol . 2001 NaN ; 185 ( 2 ): 375 – 379 . 10.1067/mob.2001.115867 11518895
Boskey ER Cone RA Whaley KJ Moench TR. Origins of vaginal acidity: High D/L lactate ratio is consistent with bacteria being the primary source . Hum Reprod . 2001 NaN ; 16 ( 9 ): 1809 – 1813 . 10.1093/humrep/16.9.1809 11527880
Cadieux P Burton J Gardiner G Braunstein I Bruce AW Kang CY Reid G. Lactobacillus strains and vaginal ecology . JAMA . 2002 NaN ; 287 ( 15 ): 1940 – 1941 . 10.1001/jama.287.15.1940
Edwards VL Smith SB McComb EJ Tamarelle J Ma B Humphrys MS Gajer P Gwilliam K Schaefer AM Lai SK The cervicovaginal microbiota-host interaction modulates Chlamydia trachomatis infection . mBio . 2019 NaN ; 10 ( 4 ): e01548 – 19 . 10.1128/mBio.01548-19 31409678
Farr A Kiss H Hagmann M Machal S Holzer I Kueronya V Husslein PW Petricevic L. Role of Lactobacillus species in the intermediate vaginal flora in early pregnancy: A retrospective cohort study . PLoS One . 2015 NaN ; 10 ( 12 ): e0144181 . 10.1371/journal.pone.0144181 26658473
France MT Ma B Gajer P Brown S Humphrys MS Holm JB Waetjen LE Brotman RM Ravel J. VALENCIA: A nearest centroid classification method for vaginal microbial communities based on composition . Microbiome . 2020 NaN ; 8 ( 1 ): 166 . 10.1186/s40168-020-00934-6 33228810
Gajer P Brotman RM Bai G Sakamoto J Schütte UM Zhong X Koenig SS Fu L Ma ZS Zhou X Temporal dynamics of the human vaginal microbiota . Sci Transl Med . 2012 NaN ; 4 ( 132 ): 132ra52 . 10.1126/scitranslmed.3003605
Galhardo CL Soares JM Jr Simões RS Haidar MA Rodrigues de Lima G Baracat EC. Estrogen effects on the vaginal pH, flora and cytology in late postmenopause after a long period without hormone therapy . Clin Exp Obstet Gynecol . 2006 ; 33 ( 2 ): 85 – 89 . 16903243
Gottlieb SL Xu F Brunham RC. Screening and treating Chlamydia trachomatis genital infection to prevent pelvic inflammatory disease: Interpretation of findings from randomized controlled trials . Sex Transm Dis . 2013 NaN ; 40 ( 2 ): 97 – 102 . 10.1097/OLQ.0b013e31827bd637 23324973
Kasprowicz A Białecka A. [Atlas of vaginal purity degrees. Research procedures, interpretation of results] (in Polish) . Wrocław (Poland) : MesPharm ; 2016 .
Kiecka A Macura B Szczepanik M. Can Lactobacillus spp. be a factor reducing the risk of miscarriage? Pol J Microbiol . 2021 NaN ; 70 ( 4 ): 431 – 446 . 10.33073/pjm-2021-043 35003275
Knezević A Stepanović S Cupić M Jevtović D Ranin J Jovanovic T. Reduced quantity and hydrogen-peroxide production of vaginal lactobacilli in HIV positive women . Biomed Pharmacother . 2005 NaN ; 59 ( 9 ): 521 – 523 . 10.1016/j.biopha.2005.06.010 16271844
Kuczyńska K. [Female vaginal biocenosis] (in Polish) . Studia Medyczne Akademii Świętokrzyskiej . 2003 ; 11 : 17 – 21 .
Kumar L Dwivedi M Jain N Shete P Solanki S Gupta R Jain A. The female reproductive tract microbiota: Friends and foe . Life . 2023 ; 13 ( 6 ): 1313 . 10.3390/life13061313 37374096
Mączyńska B. [Diagnostics of bacterial vaginosis and atypical sexually transmitted infections] (in Polish) . Diagnosta laboratoryjny . 2008 ; 6 ( 2 ): 13 – 17 .
Mei Z Li D. The role of probiotics in vaginal health . Front Cell Infect Microbiol . 2022 NaN ; 12 : 963868 . 10.3389/fcimb.2022.963868 35967876
Mendling W Brasch J Cornely OA Effendy I Friese K Ginter-Hanselmayer G Hof H Mayser P Mylonas I Ruhnke M Guideline: Vulvovaginal candidosis (AWMF 015/072), S2k (excluding chronic mucocutaneous candidosis) . Mycoses . 2015 NaN ; 58 (Suppl_1) : 1 – 15 . 10.1111/myc.12292
Mitchell CM Watson L Mitchell AJ Hyrien O Bergerat A Valint DJ Pascale A Hoffman N Srinivasan S Fredricks DN. Vaginal microbiota and mucosal immune markers in women with vulvovaginal discomfort . Sex Transm Dis . 2020 NaN ; 47 ( 4 ): 269 – 274 . 10.1097/OLQ.0000000000001143 32044865
Ness RB Hillier SL Kip KE Soper DE Stamm CA McGregor JA Bass DC Sweet RL Rice P Richter HE. Bacterial vaginosis and risk of pelvic inflammatory disease . Obstet Gynecol . 2004 NaN ; 104 ( 4 ): 761 – 769 . 10.1097/01.AOG.0000139512.37582.17 15458899
Newton ER Piper JM Shain RN Perdue ST Peairs W. Predictors of the vaginal microflora . Am J Obstet Gynecol . 2001 NaN ; 184 ( 5 ): 845 – 853 . 10.1067/mob.2001.113848 11303191
R Core Team . A language and environment for statistical computing . Vienna (Austria) : R Foundation for Statistical Computing ; 2021 [cited 2024 Jan 26 ]. Available from https://www.r-project.org
Rak K Kiecka A Białecka J Kawalec A Krzyściak P Białecka A. Retrospective analysis of the Ureaplasma spp. prevalence with reference to other genital tract infections in women of reproductive age . Pol J Microbiol . 2022 NaN ; 71 ( 4 ): 509 – 518 . 10.33073/pjm-2022-044 36401536
Ravel J Gajer P Abdo Z Schneider GM Koenig SS McCulle SL Karlebach S Gorle R Russell J Tacket CO Vaginal microbiome of reproductive-age women . Proc Natl Acad Sci USA . 2011 NaN ; 108 ( Suppl_1 ): 4680 – 4687 . 10.1073/pnas.1002611107 20534435
Ravel J Moreno I Simón C. Bacterial vaginosis and its association with infertility, endometritis, and pelvic inflammatory disease . Am J Obstet Gynecol . 2021 NaN ; 224 ( 3 ): 251 – 257 . 10.1016/j.ajog.2020.10.019 33091407
Schwebke JR Flynn MS Rivers CA. Prevalence of Gardnerella vaginalis among women with lactobacillus-predominant vaginal flora . Sex Transm Infect . 2014 NaN ; 90 ( 1 ): 61 – 63 . 10.1136/sextrans-2013-051232 24259188
Tachedjian G O’Hanlon DE Ravel J. The implausible “in vivo” role of hydrogen peroxide as an antimicrobial factor produced by vaginal microbiota . Microbiome . 2018 NaN ; 6 ( 1 ): 29 . 10.1186/s40168-018-0418-3 29409534
Vallor AC Antonio MA Hawes SE Hillier SL. Factors associated with acquisition of, or persistent colonization by, vaginal lactobacilli: Role of hydrogen peroxide production . J Infect Dis . 2001 NaN ; 184 ( 11 ): 1431 – 1436 . 10.1086/324445 11709785
Witkin SS Linhares IM. Why do lactobacilli dominate the human vaginal microbiota? BJOG . 2017 NaN ; 124 ( 4 ): 606 – 611 . 10.1111/1471-0528.14390 28224747
Witkin SS Moron AF Linhares IM Forney LJ. Influence of Lactobacillus crispatus, Lactobacillus iners and Gardnerella vaginalis on bacterial vaginal composition in pregnant women . Arch Gynecol Obstet . 2021 NaN ; 304 ( 2 ): 395 – 400 . 10.1007/s00404-021-05978-z 33521838
Zheng N Guo R Wang J Zhou W Ling Z. Contribution of Lactobacillus iners to vaginal health and diseases: A systematic review . Front Cell Infect Microbiol . 2021 NaN ; 11 : 792787 . 10.3389/fcimb.2021.792787 34881196
