Microbiome Of The Women’S Genital System

The human body is a unique living environment for many microorganisms characterized by a large diversity of species and genera. Bacteria, fungi, archaea and viruses colonizing the human body are called a microbiota [27]. They are found both on the skin surface and in the entire digestive system, from the oral cavity to the anus, in the upper respiratory tract and the urogenital system. However, most of them inhabit our digestive tract, including their greatest abundance in the large intestine, where in 1 ml of the content there are on average 10¹² microbial cells [25, 58]. Currently, the term microbiome refers to the set of genomes of archaea, commensal, symbiotic and pathogenic bacteria and fungi as well as viruses in the human environment. Clarification of this term became possible thanks to the development of molecular biology and genetic engineering, in particular metagenomic sequencing [32, 50, 58]. Acquiring knowledge of the microbiome revealed the presence of many microorganisms that had previously been unknown [27]. The first comprehensive studies of the human microbiome were launched in 2007 by implementing the international Human Microbiome Project (HMP). The project mentioned above concerned microorganisms whose discovery was based on their genomic nucleotide sequences (metagenomes), nucleotide sequences of messenger RNA (metatranscriptome), protein synthesis (metaproteome) and determination of metabolic products (metabolome) [50]. It is reported that the weight of an adult human’s microbiota is approx. 1-1.5 kg [35, 62]. Microorganisms are particularly abundant and multiply in the places of human body secretions and fluids, such as saliva, sweat, urine, tallow, runny nose, vaginal mucus and blood. Among them are microorganisms providing metabolites necessary for the proper functioning of the organism and pathogens, which cause dysbiosis in the human organism, often causing infections, inflammations, viral, bacterial or fungal diseases.

The female vagina is a characteristic microenvironment of the human body. The species diversity of the vaginal microbiota depends on many factors, such as: woman’s age, place of residence (climatic zone), quality and frequency of hygienic procedures, medications applied, e.g. antibiotics, steroids, immunosuppressants or level of hormones, mainly estrogens in the female organism [44]. The predominant group of bacteria colonizing the vagina of healthy women are the rods of Lactobacillus, while among the yeasts the most common are those of the genus Candida. Disorders of the homeostasis of the vaginal microbiota rely mainly on the change in the ratio between lactobacilli and pathogens, in favour of the latter [26, 71]. In order to restore the natural vaginal microbiota, vaginal probiotics consisting of various species of Lactobacillus, isolated from the vagina of healthy women, are used.

Until recently, it was thought that the intrauterine environment of a child’s life is sterile. However, modern microbiological and genetic tests confirm the existence of microorganisms in a child already in the prenatal period. The source of these microorganisms is the mother’s organism, and her health condition has a big impact on their quality.

During pregnancy, the female body undergoes numerous changes at the hormonal, immunological and metabolic level, necessary for the normal growth and development of the child in the prenatal period [46]. During this time, the levels of secreted hormones: progesterone and estrogens increase rapidly, and complex changes modulating the immune system, protecting the mother and child from infections are observed.

During the prenatal development of the child, the presence of bacteria has been demonstrated in the placenta, amniotic fluid and umbilical cord blood. It was found that the bacterial populations present in the placenta and amniotic fluid are similar and exhibit little diversification. The type Proteobacteria dominated and the main genera were Enterobacter, Escherichia and Shigella. Microorganisms were also detected in meconium, i.e. the first stool of newborns delivered both in the natural way and by caesarean section. There occurred mainly lactic bacteria of the genus Lactobacillus, Bifidobacterium, Leuconostoc and Enterococcus in it, as well as other ones of the genus Streptococcus, Escherichia, Enterobacter, Klebsiella and Bacteroides [38]. It is assumed that the presence of bacteria belonging to the genera: Enterocococcus, Streptococcus, Staphylococcus, Propionibacterium, Lactobacillus and Bifidobacterium in the placenta, amniotic fluid or meconium may be caused by the transition of microorganisms via the bloodstream, which is confirmed by the presence of various bacteria in umbilical cord blood [37]. In addition, in the child’s body in the prenatal period, the presence of microbiota may be the result of swallowing amniotic fluid, which affects the initial colonization of the gastrointestinal tract and skin in children, and in the case of newborn of female sex - colonization of the vagina [63].

The analysis of the composition of the bacteria species identified in meconium, placenta and amniotic fluid showed a high similarity to the species composition of bacteria found in the mouth of mothers. It has been observed that some oral cavity and gum diseases in pregnant mothers increase the risk of premature labour [1]. Therefore, it is important to care for hygiene and prevent periodontal diseases in this group of women. Another reason for the translocation of bacteria into the uterine cavity in pregnant women are prolonged vaginal infections. Aerobic, anaerobic bacteria and yeast-like fungi have also been identified in the uterine cervix. In addition to commensal bacteria of the genus Lactobacillus, pathogenic species: Enterococcus faecalis, Escherichia coli, Streptococcus agalactiae, Staphylococcus aureus, Klebsiella pneumoniae, Eubacterium lentum and yeasts Candida albicans, Candida glabrata, Candida krusei and Saccharomyces cerevisiae were the most commonly isolated [42]. Their presence in the cervical canal leads to serious gynaecological-obstetric complications, including miscarriages, premature births, infections of the amniotic fluid or lesions of the pelvic organs. Thus, intrauterine infections of the foetus and newborn may occur [12, 30, 41].

Among the postnatal factors affecting the microbiome of the newborn, the following are distinguished: the type of delivery (natural or caesarean section), diet (breastfeeding or modified milk), hygiene (sufficient or excessive) and the use or not of the antibiotic therapy or other medications in the first days of the child’s life, e.g. steroids, modifying the composition of the microflora. During natural delivery, the neonate, passing through the mother’s birth canal, has contact with the microflora derived from her vagina, mainly with the lactic fermentation rods of the genus Lactobacillus, which quickly colonise the skin, digestive tract, oral and nasopharyngeal cavity, but also in the case of the female sex, the urogenital tract [35]. That is why in the newborns of female sex, low pH of the vagina is observed during the first 2–3 weeks of life, which results from lactic acid produced by lactobacilli. Later, the pH of the vagina becomes neutral and remains so until puberty. Other bacteria acquired during physiological labour are: Bacteroides, Bifidobacterium, Enterococcus, Blautia, Veillonella, Streptococcus, Planococcus, Escherichia coli, Staphylococcus and Prevotella [20, 34, 59]. In turn, the organism of neonates born via caesarean section is initially colonized mainly by microorganisms typical of the mother’s skin and hospital strains of the genera: Staphylococcus, Streptococcus, Citrobacter, Pseudomonas, Propionibacterium, Proteus, Actinobacter, Klebsiella and E. coli [8, 20, 21, 47]. Infections with some of these bacteria are undoubtedly detrimental to the health of the child and often require applying antibiotic therapy immediately after delivery. Adverse effects of caesarean section on the risk of the occurrence of such diseases in children like: obesity, type I diabetes, celiac disease, asthma or atopy have also been observed [10, 13, 16, 43].

Over time, the differences in the microbiomes of infants born in a different way gradually diminish, however, the first microorganisms acquired during labour can persist up to approx. 6-24 month of life [8, 34, 39]. After delivery, the composition of the infant’s microbiome is shaped depending on the type of feeding. The microbiological profile of the naturally fed infant’s microbiome abounds in various microbial species, including lactic acid bacteria of the genera Lactobacillus, Bifidobacterium, Lactococcus and Enterococcus, which is not observed in artificial and mixed feeding, where a lower phylogenetic diversity occurs [47]. Madan et al. [47] observe a reduction in the number of Lactococcus bacteria in infants being exclusively breast-fed compared to those fed exclusively with modified milk. Other studies indicate that artificial feeding reduces the population size of Bifidobacterium bacteria compared to exclusive breastfeeding. However, the currently applied enrichment of dairy mixtures with prebiotics, such as short-chain galactooligosaccharides and long-chain fructooligosaccharides, increases the overall abundance of Bifidobacterium in infants fed with modified milk [55, 73]. Around the age of two, species and generic biodiversity of a child’s microbiota increases to become more and more similar to an adult human microbiota.

The mucous membrane of the female vagina is the site where numerous bacteria occur. So far, 37 genera have been identified, i.e.: Anaerococcus, Anaerotruncus, Atopobium, Arcanobacterium, Bacteroides, Bulleidia, Campylobacter, Clostridium, Coriobacterium, Corynebacterium, Dialister, Eggerthella, Enterococcus, Escherichia, Finegoldia, Gardnerella, Gemella, Klebsiella, Lactococcus, Lactobacillus, Lachnospira, Megasphaera, Mobiluncus, Mycoplasma, Parvimonas, Peptostreptococcus, Porphyromonas, Propionibacterium, Prevotella, Peptoniphilus, Rhodobaca, Rhuminococcus, Streptococcus, Sneathia, Staphylococcus, Ureaplasma, Veillonella [40, 64, 67].

The most abundant amongst them are Gram-positive lactobacilli of the genus Lactobacillus which, depending on the physiological condition of the woman, constitute 60 to even 95% of the microorganisms in the vagina of women in Europe or the United States [31]. They compete there for receptors on epithelial cells and nutrients [42]. Lactobacillus bacterium is characterized by the ability to hydrolyse glycogen, a polysaccharide found in the vaginal glands, which, as a result of their action, is hydrolysed to lactic acid. In this way, the vagina becomes acidified to a pH below 4.5, which protects the epithelium against colonization of the reproductive organs by pathogenic bacteria. Other metabolites produced by Lactobacillus bacteria are bacteriocins which exhibit bacteriostatic and bactericidal activity against pathogenic or related strains. Hydrogen peroxide is also produced, which inhibits the growth of catalase-negative and peroxidase-negative bacteria, and protease inhibitors which prevent the growth and propagation of pathogenic yeast Candida albicans.

To date, 25 species of Lactobacillus inhabiting the female vagina have been identified, i.e.: L. acidophilus, L. amylovorus, L. casei, L. crispatus, L. coleohominis, L. debrueckii subsp. bulgaricus, L. fermentum, L. frumenti, L. gasseri, L. gallinarum, L. helveticus, L. iners, L. johnsonii, L. jensenii, L. oris, L. mucoase, L.plantarum, L. paracasei, L. reuteri, L. rhamnosus, L. ruminis, L. sakei, L. salivarius, L. suntoryeus, L. vaginalis [4, 18, 76].

The structure, size and composition of vaginal microbiota depends on genetic and environmental factors and correlates with the geographical place of residence of women, individual predispositions for colonization and hormone levels [24, 63]. It is observed that the abundance of the rods of Lactobacillus is dependent on the amount of glycogen present in the vagina, which depends on the level of estrogens, female sex hormones.

Currently, many strains isolated from the vagina of women living in Europe, America, Asia and Africa have been tested (Table I). Among the species being dominant in the vaginal microbiota of Polish women, the following prevail: L. acidophilus (35%), L. fermentum (30%), L. plantarum (30%), L. delbrueckii (5%), L. rhamnosus (5%) and a small number of L. gasseri and L. casei [56, 68, 69]. In Nigeria, however, the dominant strains are: L. iners (64%), L. gasseri (8.3%), L. plantarum (7.0%), L. suntoryeus (7.0%), L. crispatus (4, 0%), L. rhamnosus (2.7%), L. vaginalis (2.7%), L.fermentum (1.3%), L. helveticus (1.3%) and L. johnsonii (1, 3%) [5].

It has also found that the microbiome of female genital tract during the nine months of pregnancy is subject to significant microbiological changes. It becomes stable and less diverse in terms of species composition, with predominance of the bacteria of the genus Lactobacillus [65]. In the examined pregnant Polish women, the most common species of Lactobacillus colonizing the vagina in the first trimester are: L. crispatus (29%), L. gasseri (19.4%) and L. rhamnosus (16.1%), in the second trimester: L. crispatus (51.6%), L. gasseri (25.8%), L. rhamnosus (19.4%) and L. amylovorus (16.1%), and in the third trimester – L. crispatus (25.8%), L. gasseri (25.8%) and L. johnsonii (19.4%) [19].

Based on the studies of the vaginal microbiota of healthy women in reproductive age from all over the world, the microorganisms occurring in the vagina are classified in V CST (Community State Types) groups [63]. The CST types distinguished: I, II, III and V, are dominated by the following lactic fermentation bacteria: L. crispatus (CST type I), L. gasseri (CST type II), L. iners (CST type III) and L. jensenii (CST type V). The CST IV type includes anaerobic microorganisms such as: Prevotella, Dialister, Atopobium, Gardnerella, Megasphaera, Peptoniphilus, Sneathia, Eggerthella, Aerococcus, Finegoldia and Mobiluncus, among which there is no specific dominant species [63, 76]. In addition, in American studies, including women from different populations, various proportions of CST types were observed depending on the ethnic group (Table II).

It was also observed that the number of Lactobacillus depends on the stage of development and life of women. Initially, in the prenatal period, the presence of various microorganisms was found, which later, in the course of the translocation of the microflora from the mouth and vagina, reach the uterus and colonize the child’s body. These include lactic bacteria of the genera: Lactobacillus, Bifidobacterium, Lactococcus and Leuconostoc [38]. After passing through the mother’s birth canal, abundant in Lactobacillus bacteria, in the female newborns, the pH of the vagina is maintained within 4.8–5.7 for 2–3 weeks, which indicates the activity of lactobacilli. However, already after 3 weeks from giving birth, the pH rises to 6–8 and persists throughout the period of childhood. During pubescence and full maturity in healthy women lactobacilli dominate during the menstrual cycle acidifying the vagina. This is due to the increasing concentration of estrogens in the follicular phase and the increase in glycogen levels. In addition, the presence of α-amylase in the vaginal mucosa supports the breakdown of glycogen into products such as maltose, maltotriose and maltotetraose, which stimulate Lactobacillus spp. to multiply and colonise [49]. However, during menstrual bleeding, the number of Lactobacillus cells decreases periodically, and the pH of the vagina increases to 7.3–7.4. During the premenopausal period, due to decreasing estrogen and glycogen levels and the vaginal mucosa becoming thin, the pH changes in the alkaline direction, and the number of lactic bacteria decreases, which promotes frequent vaginal infections caused by pathogens.

The reduction in the abundance of Lactobacillus bacteria is also influenced by steroid and immunosuppressive drugs, antibiotics and hormonal contraceptives. Steroids imitating female sex hormones, e.g. estrane derivatives, corticosteroids, adrenocortical hormones, have immunosuppressive, anti-inflammatory and antiallergic effects and contribute to reducing the number of Lactobacillus bacteria. Antibiotics, on the other hand, which act bactericidally against Grampositive, also reduce the number of lactobacilli [51]. These are the following antibiotics:

beta-lactam antibiotics: natural, isoxazole and semi-synthetic penicillins, cephalosporins, carbapenems, monobactams, macrolides, lincosamides, fusidic acid;

The vaginal microbiota is also influenced by the use of hormonal contraceptives, containing low doses of estrogen, which blocks ovulation and maturation of the endometrium. Thus, the level of glycogen in the vagina is low, resulting in a reduction in the number of Lactobacillus rods. The result thereof are frequent infections of the female vagina: bacterial vaginosis (BV) and vaginal candidiasis.

The term mycobiome is defined as the sum of genomes and genes belonging to the fungi which inhabit a specific biological niches. The female genital tract, and in particular the vagina, is a specific microenvironment for fungi, to which considerable attention has been paid in the last few years. Molecular technique – NGS sequencing (Next Generation Sequencing) and bioinformatics tools play an important role in understanding the diversity of fungi and are useful in identifying microorganisms and providing a new insight into fungal ecology. Initial studies were based on the techniques of culturing the material obtained through swabs from healthy volunteers or diabetics, teenagers or pregnant women. Fungi were detected in 20–60% of trials [11]. Among them, the yeasts of the genera Candida and Saccharomyces dominated, to a lesser extent there occurred filamentous fungi of the genera Aspergillus, Alternaria and Cladosporium. The presence of these microorganisms depends on the age, hormonal cycle, female health status and the presence of bacteria in the genital tract and specific vaginal environment [11, 14, 50]. Without exception, the dominant member of the fungal community is Candida albicans (85–95% of the Candida population), although there are other species of this genus, e.g. C. krusei, C. parapsilosis, C. tropicalis, C. glabrata, C. guilliermondii, C. pseudotropicalis, C. stellatoidea and others [11, 66]. Guo et al. [29] using the molecular method for identifying the 18S rRNA gene, identified 3 families of fungi: Ascomycota, with the dominant genus of Candida, and Basidiomycota and Oomycota. They also established that women suffering from allergic rhinitis or recurrent vaginal candidiasis had a higher percentage of C. albicans, and lower percentage of S. cerevisiae or other unidentified fungi in comparison with healthy women. In turn, Drell et al. [22], by sequencing a ITS1 fragment of DNA in samples obtained from the vaginas of 294 healthy women in Estonia, established the presence of Ascomycota in 58% of trials, and Basidiomycota in only 3% of trials. Within the type Ascomycetes the researchers have detected representatives of the orders Saccharomycetales (mainly the genus Candida), Capnodiales, Eurotiales, Pleosporales and Helotiales. C. albicans was detected in 68% of sequenced samples, while many sequences were not systematically assigned due to limited resources of available databases connected with sequencing fungi. On the other hand, in the studies by Bitew and Abebaw [9] in samples of swabs obtained from 210 women in Ethiopia, the following yeasts were identified C. albicans in 58.6% of isolates, C. krusei in 17.2%, C. dubliniensis in 9.2%, C. glabrata in 3.4 % and C. inconspicua, C. tropicalis, C. kefir, C. guiiliermondii, C. lusitaniae and C. parapsilosis in individual cases. The researchers used an automated, compact Vitek 2 system for identification, based on fluorescent detection of yeast growth and metabolic changes occurring in microwells of special cards placed in an incubator connected to the reader [9]. Similar results were obtained in the study of vaginal swabs from women in Saudi Arabia, using various identification methods, where the dominant species in both diseased and healthy women was C. albicans, followed by C. dublinienesis and C. glabrata; while C. tropicalis was found only in diseased women [2]. In turn, in studies conducted in Egypt in 62.7% of samples of swabs from women with symptoms of candidiasis Candida yeasts were found, of which 81.1% were C. albicans, 8.1% C. glabrata and C. tropicalis and 2.7% S. cerevisiae [66].

Yeasts enter the human organism from the natural environment and can be transferred to newborns during labour, colonizing specific human body niches. Ward et al. [72] conducted research on the development of mycobiome in infants in the first months of life, depending on the method of delivery. They found that dominant species in samples obtained from various parts of the body of infants (skin, mouth, anus) were C. parapsilosis, C. tropicalis, S. cerevisiae, C. albicans, C. othopsilosis, Cryptococcus pseudolongus, Cladosporium velox, Debaryomyces rhenium, D. hansenii, Hanseniaspora uvarum, and C. krusei. On the other hand, the composition of the vaginal mycobiome of the women giving birth was dominated by C. albicans, while in the mycobiome of the anus C. albicans, S. cerevisiae, and C. parapsilosis were the most numerous. The overall numbers of detected taxa were similar in the samples obtained from infants and women. Researchers established that the way the children were delivered had an effect on the number of species, but not on the structure of the fungal population. Naturally born infants did not exhibit a greater similarity of the skin mycobiome to their mother’s vaginal mycobiome than the neonates born by caesarean section. However, the difference was visible in the number of C. albicans, which occurred in a significantly higher number on the skin of 1-month-old infants born naturally in comparison to the infants born by caesarean section [72].

There is more and more evidence indicating that estrogens have a positive effect on the colonization of the vaginal mucosa and subsequent infection caused by Candida yeast. Some Candida species possess estrogen receptors. It has been demonstrated that estrogen interferes with the chemotaxis of neutrophils to the vaginal epithelium and inhibits Th17 lymphocytes differentiation, which consequently can lead to an increased susceptibility of the host to the pathogens of the genus Candida. Some reports link symptomatic vaginosis to the luteal phase just before menstruation, when high estrogen level is observed, as well as an increase in vaginal pH level, which additionally facilitates its colonization by the pathogenic yeasts. It has also been proven that Candida can metabolise lactate derived from lactic acid produced by lactic bacteria in the vagina and it has been shown that in the presence of lactic acid C. albicans is less efficiently attacked by macrophages and can change the profiles of immune cell cytokines by raising the production of interleukin 10 (IL-10) and lowering IL-17. Studies confirm that Candida can evolve to stop the immune response and increase the area of its commensalism.

The initial adhesion of yeast to epithelial cells is regulated by hydrophobic interactions between surfaces. The attachment of yeast to the epithelial cell receptors is mediated by adhesion proteins [11, 14]. High capacity of yeasts for adhesion to epithelial cells obtained from women suffering from recurrent candidiasis of vulva and vagina (VVC – VulvoVaginal Candidiasis) has been demonstrated. It is estimated that 3 out of 4 women have been infected with candidiasis at least once in their lifetime, and 20% of healthy women may also be asymptomatic carriers that have been infected with this microorganism in commensal interactions. There exists a view whereby the predispositions for being colonised by Candida are niche-dependent, but they also depend on host immune defects, interrupted vaginal epithelium and microbiological dysbiosis. A statistically significant increase in the number of C. albicans pathogenic yeasts is observed in women with VVC compared to the number of these microorganisms in healthy women, while no significant differences have been observed in the number of Candida yeasts not belonging to the albicans species. These observations confirm the theory of “fungal burden threshold”, above which inflammatory cells occur, whose metabolites are the direct cause of the symptoms of vaginal infection, such as itching, irritation, burning and the appearance of secretions. Candida is a polymorphic fungus in which morphogenic transitions are also part of the pathogenicity mechanism. The yeast form is associated with asymptomatic colonization, transmission and spreading especially through the circulatory system, while the mycelial form is associated with adhesion and mucosal invasion, characteristic for disease symptoms, and the production of agents that cause macrophage lysis [11]. Morphogenesis, however, depends on various factors, including the activity of macrophages, the presence of bacteria and their metabolites in the vagina and estrogen levels [14].

C. albicans virulence factors include extracellular hydrolytic enzymes, heat shock proteins and factors leading to biofilm production. In vitro studies of the proteolytic activity of C. albicans isolates from women with VVC have shown increased activity of enzymes, especially from the aspartic protease family, which are virulence factors disturbing the continuity of the mucosa and inducing immunopathology. Yeast also produces several extracellular enzymes, such as phospholipases, lipases and haemolysins, essential for tissue invasion and nutrient uptake, especially iron released from haemoglobin degradation [14]. Another hypothesis on the pathogenicity of C. albicans is associated with the secretion of toxins. Moyes et al. [54], using a mouse model of oropharyngeal candidiasis, identified a cytolytic peptide toxin, candidalysin, essential for the pathogenesis of the mucosa. Another pathogenic feature of C. albicans is the ability to create a biofilm in contact with, e.g. mucous membranes, followed by the production of hyphae capable of penetrating host tissues. C. albicans biofilm is formed by polysaccharides such as α-mannan, α-1,6 glucan and α-1,3 glucan. The ability to form biofilms is also demonstrated by C. glabrata, C. guilliermondii, C. parapsilosis, and C. tropicalis. All of the above species are capable of creating an invasive hyphal form. In the context of triggering candidiasis, biofilms have been reported to be up to 1000-fold more resistant to antifungal agents used in the treatment, compared to the cells that do not form biofilms [14].

An important issue is an interaction between the bacteria found in the vagina, mainly the rods of Lactobacillus and the pathogenic Candida yeasts. In general, there are 5 ways of fungi-bacteria interaction that can also be present in the vagina, i.e. physical interactions, chemical changes, metabolic by-products, environmental changes and changes in the host’s immune resistance [61]. The most reliable hypothesis regarding these interactions is that the bacteria found in the vagina do not prevent the mucosa colonizing from Candida, but only limit the excessive growth of yeasts, among others through the production of lactate and short-chain fatty acids inhibiting the passage of the pathogen from the cellular stage to the hyphal one [11]. In addition, women with VVC have a less abundant Lactobacillus population. An additional factor influencing the disruption of balance between bacteria and yeasts is antibiotic therapy, but no unequivocal correlation between taking antibiotics and the incidence of VVC has been proven [11]. Another hypothesis is that the hydrogen peroxide secreted by Lactobacillus bacteria is involved in the prevention of adhesion to epithelial cells and is responsible for the control of Candida growth, in addition to other mechanisms associated with the activity of lactobacilli which are not fully understood yet [14]. Mutual interactions of these microbial groups are also dependent on the formation of a multispecies biofilm on the mucous membrane surface. In vitro studies have confirmed the negative impact of Lactobacillus on the formation of mycelium and the suppression of genes responsible for this process [11]. Research into mutual interactions of lactic acid bacteria and yeasts is still continuing and very much needed in the context of VVC prevention and treatment.

Characteristics of the human mycobiome is important for the development of diagnostics and preventive measures in fungal diseases, including vaginal candidiasis.

Female vaginal microbiota disorders are most often caused by a decrease in the number of lactobacilli to the advantage of pathogenic microorganisms. This results in various types of infections, i.e. bacterial vaginosis and vaginal candidiasis [7, 57, 60]. Vaginal homeostasis disorders are caused by antibiotics, steroid and immunosuppressive drugs, as well as hormonal and barrier contraceptive agents such as condoms, caps and vaginal diaphragms which damages the vaginal epithelium. Other factors with poorly understood mechanism of influencing the vaginal microbiota are: poor hygiene, use of underwear made of synthetic materials, vaginal irrigation, weakening of the immune system as a result of systemic infection or chronic diseases or stress, use of tampons, sexual intercourse or even swimming in a pool during which some rods of Lactobacillus may be partially washed out [50]. Therefore, it is important to quickly restore the original state of the microbiota in this part of the female body by using live cultures of lactic acid bacteria in the form of vaginal probiotics. These are microorganisms which favourably influence the vaginal microbiota by combating the pathogens which are present there. They should meet the criteria set in 2001 and 2002 by the European Food Safety Authority (EFSA) and the World Health Organization (WHO) [28].

Among the basic requirements for vaginal probiotics the following are listed:

being derived from the urogenital system of young women;

It has been demonstrated that the use of the L. acidophilus probiotic strains for 6–12 days and L. rhamnosus GR-1, L. fermentum RC-1 for 2 months inhibits the growth of the bacteria causing BV. This is the result of producing hydrogen peroxide, lactic acid, bacteriocins and the inhibition of the adherence of G. vaginalis [23].

Polish studies have demonstrated that oral administration of three specific strains: L. gasseri, L. fermentum and L. plantarum to women of reproductive age, increased the number of Lactobacillus rods in the anus and vagina of the patients. At the same time, a decrease in vaginal pH and an effective change in the Nugent scale values has been observed, which is used to assess the vaginal biocenosis based on proportional shares of particular types of bacteria in the preparation tested. However, the use of probiotic is not always effective, especially when the urogenital tract infection is very advanced [70]. In addition, it was found that other species of Lactobacillus, which are not the natural, native microbiota of the vaginal epithelium, cannot colonize intimate areas permanently. They occur only locally during the application of the probiotic, and after the end of supplementation they die and are excreted from the body. According to the data published in the journal Nature [48], the immune system ultimately determines what the quantitative and qualitative composition of the microbiota of our body will be. This is confirmed by the studies in which a vaginal probiotic containing L. rhamnosus strain was used by women who live in the United States. On the third day of the experiment, the strain was characterized by a high titre but on the 21st day it was barely detectable. The supplemented microorganism was not identified as a typical, native microbiota of the genital tract of American women. It was only a temporary colonizer. The authors suggest that the elimination of the L. rhamnosus probiotic strain occurred due to the natural self-regulating mechanisms of the vaginal ecosystem [3].

Vaginal probiotics available on the pharmaceutical market are in the form of freeze-dried cellular biomass immobilized in hard cellulose or gelatine capsules. The frequency of their application depends on the severity of infection resulting from vaginal microbiota disorders. They can colonize the vagina or stay there long enough to cause the restoration of native, natural microbiota. For this purpose, they must produce L (+) lactic acid in an amount of more than 50% of the total amount of acid produced and possibly other organic acids, such as acetic, formic, propionic, benzoic or mevalonic acid. They should have the ability to adhere to the vaginal epithelium, which requires testing their hydrophobic properties, surface charge, surface structure and affinity for the epithelial cell receptors.

Knowledge and understanding of the functioning of the female reproductive system microbiota is important in the treatment and prevention of vaginal infection. The more so because they are often chronic and difficult to treat, which constitutes a contemporary challenge in urology and gynaecology. In particular, vaginosis and candidiasis which occur in pregnant women require treatment due to the risk of miscarriages and intrauterine infections. More and more knowledge about the role of probiotic bacteria in the treatment of vaginal infections allows the search for new, effective strains. It is important that they have GRAS (generally recognized as safe) status, i.e. they are non-pathogenic and do not produce toxic, carcinogenic, teratogenic and mutagenic substances. In addition, they should survive the technological process and storage period in dry and refrigerated conditions for 12 to 24 months. All the more so because during production, the bacteria are most often subjected to the freeze-drying process (lyophilization), which allows for obtaining permanent preparations which preserve their viability. In addition, a probiotic preparation which is a medicine should be manufactured in accordance with good manufacturing practice (GMP) and be appropriately labelled. There is a great need to compose new vaginal probiotics on the Polish market, which is deficient in preparations based on native Lactobacillus bacteria strains.