The dynamics of vaginal and rectal Lactobacillus spp. flora in subsequent trimesters of pregnancy in healthy Polish women, assessed using the Sanger sequencing method

The genus Lactobacillus, containing over 180 species, is composed of Gram-negative bacilli, which prefer microaerophilic or strictly anaerobic conditions. They form part of the human microbiome, as they colonize the lower part of the digestive tract, vagina and mouth. They are of particular importance in the female genital tract, where due to lactic acid synthesis, they reduce the pH, and therefore, protect it from bacterial or fungal infections. These bacteria also produce bacteriostatic and bactericidal substances, such as bacteriocins or hydrogen peroxide (H₂O₂), thereby eliminating the competing species in their environment [1, 2]. A reduction in the load of bacteria from the genus Lactobacillus with a simultaneous increase in the number of pathogenic anaerobic bacteria, such as Gardnerella vaginalis, Prevotella spp., or Atopobium vaginalis, is the main cause of bacterial vaginosis (BV) [3‐5]. Maintaining the microbial balance of the vagina is of the greatest importance in the prevention of bacterial vaginosis; hence, in recent years, pharmaceuticals and dietary supplements containing probiotic strains of the genus Lactobacillus have been growing in popularity [6].

The microbial species composition of the vagina changes in individual periods of a woman’s life and depends on hormonal changes. In the course of fetal life, the reproductive system is devoid of microorganisms. During delivery, the newborn acquires the mother’s microbial flora and is under the influence of maternal estrogens in the first months of life. In infancy and early childhood, as a result of withdrawal from maternal hormones, bacteria of the genus Lactobacillus are completely absent, and the pH ranges from 6.0–8.0. They reappear when a woman enters puberty, together with the first menstruation. Shifts in the microbial flora of the vagina depend on hormonal changes. In the first stage of the menstrual cycle, when the body is under the control of estrogens, the number of bacteria from the genus Lactobacillus increases, while in the second phase of the cycle, as a result of progesterone dominance, the vaginal pH rises and the numbers of Lactobacillus fall. Changes in the vaginal flora also depend on other factors, such as: antibiotic therapy, hormone therapy, oral contraception or pregnancy [7].

During pregnancy, there are numerous hormonal changes in the woman’s body. As the pregnancy progresses, progesterone, prolactin and estrogen levels rise. There is also an increase in the concentration of thyroxine, a thyroid hormone, secreted. Additionally, the woman is under the influence of placental hormones, such as: placental gonadotropin, human placental lactogen, corticotropin, placental tyreotropin and steroid hormones [8]. Hormonal, as well as immunological and metabolic, changes in pregnancy significantly affect the microbiome composition [9‐11].

Vaginal microbiota is dominated by bacteria from the genus Lactobacillus [12]. During pregnancy, significant modifications are also present in this niche. Bacterial diversity is reduced, while species stability and the number of species from the genus Lactobacillus are increased [12]. The predominant species among Asian and Caucasian pregnant women is L. crispatus, while L. gasseri is prevalent in Afro-Americans [13, 14].

Phenotypic methods used for species identification for the genus Lactobacillus are insufficient, therefore, molecular methods are currently most often employed. One such method is Sanger sequencing, which allows identification of species through analysis of the 16 s rDNA conservative sequence [15]. This technique is characterized by high sensitivity and specificity and makes it possible to obtain unambiguous and repeatable results [16].

Currently, there is a lack of data concerning the bacterial species composition as regards the genus Lactobacillus colonizing the vagina and rectum of pregnant women in Poland. Therefore, the objective of the present study is to analyze the dynamics of vaginal and rectal microbiota composition, formed by the species from the genus Lactobacillus in the three subsequent trimesters of pregnancy, which were identified using the Sanger molecular sequencing method. In these studies, we first determine the species composition of the Lactobacillus genus in the vagina and rectum, and then the examined effect of the trimesters of pregnancy on changes in the dynamics of colonization by lactobacilli. Research conducted on a healthy pregnant population may be a starting point for studies in groups of women with a specific underlying disease, e.g. diabetes.

The employment of Sanger sequencing allowed identification of 15 different species of Lactobacillus which colonize the vagina at least once during pregnancy in tested population. In the first trimester, 12 species were determined, in the second trimester: 11, and in the third: 9. A qualitative analysis of the subsequent trimesters demonstrated that, in the first trimester, the vaginal species most commonly isolated were: L. crispatus 29% (n = 9), followed by L. gasseri 19.4% (n = 6), L. rhamnosus 16.1% (n = 5), L. jensenii 12.9% (n = 4), L. johnsonii 12.9% (n = 4), L. amylovorus 9.7% (n = 3), L. helveticus 6.5% (n = 2), L. reuteri 6.5% (n = 2), L. casei 3.2% (n = 1), L. plantarum 3.2% (n = 1), L. salivarius 3.2% (n = 1) and L. vaginalis 3.2% (n = 1). On the other hand, in the second trimester, the most often present species were as follows: L. crispatus 51.6% (n = 16), L. gasseri 25.8% (n = 8), L. rhamnosus 19.4% (n = 6), L. amylovorus 16.1% (n = 5), L. jensenii 9.7% (n = 3), L. antri 6.5% (n = 2), L. reuteri 6.5% (n = 2), L. coleohominis 3.2% (n = 1), L. helveticus 3.2% (n = 1), L. johnsonii 3.2% (n = 1) and L. salivarius 3.2% (n = 1). While in the third trimester, the most frequent species were: L. crispatus 25.8% (n = 8), L. gasseri 25.8% (n = 8), followed by L. johnsonii 19.4% (n = 6), L. rhamnosus 16.1% (n = 5), L. jensenii 9.7% (n = 3), L. reuteri 9.7% (n = 3), L. amylovorus 6.5% (n = 2), L. antri 3.2% (n = 1) and L. delbrueckii subsp. bulgaricus 3.2% (n = 1) (Fig. 1).

Analysis of the numbers of Lactobacillus [CFU/ml] colonizing the vagina in the three subsequent trimesters of pregnancy demonstrated that the most bacteria could be found in the first trimester, 1.10E + 06 CFU/ml, on average, while in the ensuing trimesters the average number was 4.30E + 05 CFU/ml in the second one and 4.19E + 05 CFU/ml in the third. Detailed results are presented in Table 1, Fig. 2a, b and c.

In the group of women studied, during the entire pregnancy period, the vagina was colonized by 5 species from the genus Lactobacillus in 9.7% (n = 3) of the patients; in 3.2% (n- = 1) of patients there were 4 species present, and most often, because in as many as 32.3% (n = 10) of patients, the presence of 3 various species was recorded; in 29% (n = 9) of patients there were two species, while in 16.1% of women (n = 5) there was one species present. In 9.7% (n = 3) of the patients, throughout their pregnancies, there were no Lactobacillus species determined.

Analysis of the dynamics of the percentage share of the Lactobacillus species in the microbiota composition showed differences in the general and individual colonization of the genital tract in pregnant women. In the group of patients subjected to the study, 58% were colonized in each trimester with at least one Lactobacillus species, in 19%, Lactobacillus was present in two successive trimesters. In 6.5% of women, Lactobacillus colonized the vagina in the first trimester. Similarly, in 6.5% of women, Lactobacillus was present in only one trimester. However, these differences were not statistically significant, which may stem from the fact that the studied group of patients was too small.

Analysis of the frequency of occurrence of particular species colonizing the vagina in individual patients showed diversity. In 9.7% of the patients, L. crispatus was present throughout the pregnancy, in 22.6% of women this species was identified in two subsequent trimesters, while in 6.5% of the cases, L. crispatus appeared in the first and third trimesters. L. gasseri, in the case of one patient (3.2%), was present in the vagina throughout the entire pregnancy, three patients (9.7%) were colonized in two successive trimesters, and two others (6.5%) in the first and third trimesters. In two women (6.5%), L. rhamnosus colonized the vagina in the three trimesters of gestation, while two others (6.5%) were colonized by this species in two subsequent trimesters. In three women (9.7%), L. jensenii was present in two subsequent trimesters, while L. johnsonii was identified in the first and third trimesters in two patients (6.5%). L. reuteri colonized the vagina for two subsequent trimesters in two patients (6.5%), and in one (3.2%), it appeared in the first and the last trimesters. L. amylovorus and L. antri were isolated in two consecutive trimesters in 3.2% of the studied patients. L. casei, L. delbrueckii sb.bulgaricus, L. helveticus, L. salivarius, L. vaginalis, L. coleohominis and L. plantarum were found only in one trimester.

In the rectal isolate pool studied, 17 different species from the genus Lactobacillus that appeared at least once throughout the entire period of pregnancy were identified. The most, as many as 14, species colonized the rectum in the first trimester; in consecutive trimesters, this number decreased by around 50%: in the second trimester, 8 species were identified, and in the third, there were only 7 (p = 0.003).

Analysis of the dynamics of the species composition in the rectum in the three subsequent trimesters of pregnancy demonstrated that, in the first trimester, the most frequently encountered species were the following: L. johnsonii 19.4% (n = 6), L. plantarum 9.7% (n = 3), L. casei 6.5% (n = 2), L. crispatus 6.5% (n = 2), L. jensenii 6.5% (n = 2), L. ruminis 6.5% (n = 2), L. salivarius 6.5% (n = 2), L. acidophilus 3.2% (n = 1), L. amylovorus 3.2% (n = 1), L. frumenti 3.2% (n = 1), L. gasseri 3.2% (n = 1), L. mucosae 3.2% (n = 1), L. paracasei 3.2% (n = 1) and L. rhamnosus 3.2% (n = 1). In the second trimester, the most frequent species was L. crispatus 9.7% (n = 3), followed by L. mucosae 6.5% (n = 2), L. casei 3.2% (n = 1), L. gasseri 3.2% (n = 1), L. johnsonii 3.2% (n = 1), L. plantarum 9.1% (n = 1), L. reuteri 9.1% (n = 1), L. salivarius 9.1% (n = 1). On the other hand, in the third trimester, the species isolated most often were: L. casei 9.7% (n = 3), L. rhamnosus 9.7% (n = 3) and L. crispatus 6.5% (n = 2) and then L. jensenii 3.2% (n = 1), L. ruminis 3.2% (n = 1), L. sakei 3.2% (n = 1), L. salivarius 3.2% (n = 1) (Fig. 3). Statistically significant differences as regards the incidence was shown for the L. johnsonii (p = 0.014) species. Analysis of the numbers of [CFU/ml] Lactobacillus in the three subsequent trimesters, similarly as in the case of the vagina, showed a slight decrease in the numbers in successive trimesters. In the first trimester, the average numbers of the Lactobacillus bacteria was 1.06E + 05 CFU/ml, while in the second trimester it was 1.50E + 04 CFU/ml, and in the third: 7.43E + 04 CFU/ml. Detailed results are shown in Table 1, Fig. 4a, b and c. The analysis of the numbers of individual species demonstrated significant differences in the case of L. johnsonii (p = 0.003).

In the group of women studied, the most species of Lactobacillus, because it was as many as five different ones, were isolated from only one patient (3.2%); in three patients (9.7%), three different Lactobacillus species were isolated, and in nine (29%) women, two various species were isolated, while in ten (32.3%) women, one species was present throughout their pregnancies. In eight patients (25.8%), there was no determination of bacteria from this species in the rectum throughout the gestation.

The studied pool of all Lactobacillus strains, isolated from both the vagina and anus, were also described in terms of their ability to produce H₂O₂. It was demonstrated that 84% of all the isolates examined were H2O2-positive and 16% were H2O2-negative. Among the H2O2-producing species, there were: L. acidophilus, L. casei, L. crispatus, L. delbrueckii subsp. bulgaricus, L. gasseri, L. helveticus, L. jensenii, L. johnsonii and L. reuteri.

The Sanger method has found broad application in species identification as regards the Lactobacillus strains, both in science [16, 20], and the food industry, particularly in typing species responsible for fermentation of dairy products [21].

Based on this method, this study has identified 15 various species of Lactobacillus colonizing the female reproductive tract. The same outcome was obtained by Anderson et al., who also identified 15 Lactobacillus species colonizing the vagina in the group of German patients studied [16]. Despite the fact that the qualitative and quantitative composition of bacteria from the genus Lactobacillus depends on numerous factors, as it is clear from literature data, the species that most often colonize the reproductive tract in healthy women are L. crispatus, L. jensenii and L. gasseri [22]. These observations coincide with the results of our studies, which demonstrate that the dominant species was L. crispatus, while L. gasseri was the second most commonly isolated species colonizing the vagina in each of the three trimesters of gestation. Our results correspond with studies conducted on Polish women by Martirosian et al. [23], who compared the vaginal flora of healthy women and patients after a kidney transplantation. She identified two species in healthy women, which were L. crispatus and L. gasseri, however two species remained unidentified. This might be caused by different methodology that was based on the PCR reaction. Comparable results were also described by Pendharkar et al., who examined the microbiome in women living in South Africa. They discovered the predominance of L. crispatus, L. iners, L. jensenii, L. gasseri and L. vaginalis [5]. On the other hand, investigations carried out by Alioua et al., aiming at comparing the microbiological flora of the reproductive tract in healthy women and those with a diagnosis of vaginosis, found the presence of the following species: L. crispatus, L. jensenii, L. johnsonii, L. acidophilus, L. iners and L.delbrueckii in the female vagina which, according to the Nugent score, were classified as being below 2.66 [24]. Examinations conducted by Korean scientists, using qPCR, also reflected the results of analyses presented in this study. They showed that, in the group of patients studied, the species isolated most often were L. crispatus, L. gasseri, L. jensenii, L. vaginalis and L. iners [25].

Analysis of the species dynamics for individual patients demonstrated that 32.3% of women were colonized with three various species of Lactobacillus for the entire pregnancy, and a similar finding was obtained by Petricevic et al. [14], who demonstrated the presence of three species in 39% of their pregnant patients. Likewise, in the case of colonization with four species, in which Petricevic found their presence in 4% of the patients, our testing gave a result of 3.2%.

Characterization of the species composition in the rectum found a greater number of species in comparison with the vaginal flora. In this examination, 17 various Lactobacillus species were identified, however, their diversity in individual trimesters of gestation was smaller than in the case of the vagina. In the patient group studied, 16.1% were colonized in the rectum with bacteria from the genus Lactobacillus throughout their pregnancies, however, the species composition was subject to significant changes (p = 0.003), as it was demonstrated in the analysis, none of the patients was colonized by the same species in the three consecutive trimesters. The majority of the species appeared only once during the entire pregnancy. The exceptions were species such as L. crispatus, which in the case of two patients (6.5%), was present in two subsequent trimesters, and in one (3.2%), in the first and last trimester. In 3.2% of the patients, L. mucosae was isolated in the first and second trimesters of gestation, while L. ruminis was found in the first and third trimesters. We presume that the decrease in the microorganisms in the rectal sample during three trimesters might be caused by physiological changes in woman’s body, especially in the third trimester, such as bloating and hemorrhoids, which can affect the imbalance in this niche, as well as impede proper material collection. We believe this should be verified in the extended patient group.

The analysis of the species composition of Lactobacillus colonizing the rectum in pregnant women showed that, out of the 17 identified species, the predominant ones were L. crispatus, L. casei and L. johnsonii. The finding obtained is confirmed by the work by Song et al., who using the multiplex PCR reaction, identified the following species in feces: L. acidophilus, L. crispatus, L. fermentum, L. gasseri, L. jensenii, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius, L. paracasei and L. delbrueckii [26]. While Cooperstock et al., when studying the feces of Europeans, both neonates and adults, demonstrated the presence of species such as L. salivarius, L. fermentum and Lactobacillus species belonging to the acidophilus group (among others, L. crispatus, L. gasseri, L. johnsonii, L. amylovorus) [27]. Similar findings were presented by a Swedish scientific team, according to which, the species most frequently isolated from the anus were: L. plantarum, L. rhamnosus and L. paracasei [28].

In our paper, we had characterized the dynamics of the Lactobacillus species in three subsequent trimesters in 32 healthy pregnant Polish women. We believe that due to the lack of data from our geographical region, the strengths of our study lie in testing this particular group of patients, the long testing duration of individual patients, and the applied methodology, which included the quantitative cultivation of the collected material and species identification by the Sanger sequencing method. Nevertheless, some limitation of our work can be observed in the lack of a correspondingly large group of patients, which would make it possible to determine more statistically significant differences. To improve the statistical aspect of our project, an extension of the study group could constitute a starting point for further investigation, which compares Lactobacillus flora in healthy women in physiological pregnancy to patients with a pathology of pregnancy, such as gestational diabetes, endocrine and immunological disorders.

In the present study, we have demonstrated that the microbiota consisting of the Lactobacillus species in the vagina in pregnant women is stable, both quantitatively and qualitatively, in the three subsequent trimesters of gestation. While in the rectum, the numbers of Lactobacillus species in the first trimester is significantly higher. Additionally, we have shown that the dominant vaginal species is L. crispatus, while the prevalent rectal species is L. johnsonii. The species composition was subject to change in the case of both individual patients, as well as in the whole group subjected to the study; however, these differences were not statistically significant.