Phascinating Phages

Review

Marek Straka 1,2, Martina Dubinová 1,3 and Adriána Liptáková 1

1 Medical Faculty, Institute of Microbiology, Comenius University in Bratislava, 81108 Bratislava, Slovakia
2 Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia
3 St. Elizabeth University of Health and Social Science, 81102 Bratislava, Slovakia

Microorganisms 10, 1365, 2022
https://doi.org/10.3390/
microorganisms10071365

Treatment of infections caused by bacteria has become more complex due to the increasing number of bacterial strains that are resistant to conventional antimicrobial therapy. A highly promising alternative appears to be bacteriophage (phage) therapy, in which natural predators of bacteria, bacteriophages, play a role. Although these viruses were first discovered in 1917, the development of phage therapy was impacted by the discovery of antibiotics, which spread more quickly and effectively in medical practice. Despite this, phage therapy has a long history in Eastern Europe; however, Western countries are currently striving to reintroduce phage therapy as a tool in the fight against diseases caused by drug-resistant bacteria. This review describes phage biology, bacterial and phage competition mechanisms, and the benefits and drawbacks of phage therapy. The results of various laboratory experiments, and clinical cases where phage therapy was administered, are described.

Keywords: bacteriophages; drug-resistant bacteria; phage therapy; antibiotic therapy

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1. Introduction

In recent decades, an increasing number of antibiotic-resistant bacterial strains have been reported. Therefore, conventional antibiotic therapy is often ineffective [1,2,3], leading to the search for new treatment options for bacterial infectious diseases. Several possibilities exist to overcome bacterial resistance to antibiotics; for example, the development of new effective antibiotics [4]; the testing of the antimicrobial effect of natural substances, such as herbal products [5,6], honey [7] or wine products [8]; the use of photodynamic inactivation [9] or the quorum-quenching approach [10]; or therapy that modulates the patient’s own immunity using immunomodulators of microbial origin or therapeutic autovaccines prepared from the patient’s own strains [11]. One of the most promising alternatives to antibiotic treatment is bacteriophage therapy, which uses natural enemies of bacteria, bacteriophages [12]. Bacteriophages, or phages, are viruses that control the growth and spread of their bacterial hosts. They are the most widespread entity in the biosphere and are found wherever bacteria live, for example, in salt waters, cold waters, hot springs, waste waters, or sewage [13,14,15]. They are also the most abundant component of the human microbiome, playing an important role in intestinal microbial composition and horizontal gene transfer [16,17,18]. Due to their unique life cycle and biological properties, phages are a potential powerful weapon in the treatment of bacterial infections. These properties, the differences between antibiotics and phages, and the developments in phage therapy are discussed in the following sections.
 

2. Phage Biology

2.1. Phage Life Cycle

The first step during phage infection is adsorption of phages to the surface structures of bacteria by tail fibers or bacteriophage spikes [14]. Phages are capable of infecting only those bacteria that have the corresponding receptor, which determines the host spectrum of the phages. This spectrum is also limited by the defense mechanisms of bacteria against phages [18]. This interaction results in a different host specificity—some phages are strain-specific, whereas others are able to infect a wider range of bacterial species or even genera [19].
After successful adsorption, a pore is formed in the membrane of the infected bacterium and the phage genome is subsequently injected into its cytoplasm [13]. Phage replication may be performed in two basic ways [19,20]. In the lytic cycle, phages exploit the proteosynthetic apparatus of bacteria to synthesize their proteins, replicate phage nucleic acid (NA), assemble phage particles, and release themselves from the cell while disrupting the host bacterial cell [14]. During the release, phages use holins to perforate the cytoplasmic membrane and endolysins to destroy the cell wall membrane, thus lysing the bacterial cell [21].
In the lysogenic cycle, the phage genome is integrated into the bacterial chromosome in the form of a prophage. Such an integrated phage can persist in the genetic material of the bacterium for a long time, and replicate its nucleic acid together with the replication of the bacterial chromosome. Prophages may persist in this state until they switch to the lytic cycle [20]. Lysogeny may provide benefits to the bacterial cell because phages are able to construct gene-encoding products that are involved in antibiotic resistance or virulence factors that enhance the infectious process (e.g., genes for diphtheria toxin [22] or Panton–Valentin leukocidin [23]). Therefore, in the context of phage therapy, a lytic life cycle is desirable, in addition to the absence of genetic determinants of antimicrobial resistance and bacterial virulence in the genome of therapeutic phages [15]. Differences between the lytic and lysogenic life cycle of bacteriophages are shown in Figure 1.
Microorganisms 10 01365 g001 550
Figure 1. Life cycle of bacteriophages. First, the phage adsorbs to the bacterial cell receptor (1). Second, it forms a pore in the membrane and phage NA is injected into the host cell (2). As the virus continues the lytic cycle, viral proteins are produced, phage NA (3a) replicates, and virion assembly (4a) and cell lysis (5a) occur. During the lysogenic cycle, the phage integrates its genome into the host chromosome (3b) and replicates with it (4b). Changing conditions may induce a transition to the lytic cycle.

2.2. Phages versus Bacteria

During evolution, bacterial strains developed mechanisms to protect themselves from phage infection. One of these mechanisms is superinfection exclusion. In this case, the lysogenized bacterium that carries a prophage in its genome cannot be infected by another phage. Other mechanisms include preventing phage adsorption to the bacterial cell surface due to alteration of the receptor [18] or the production of protective surface polysaccharides that mask the receptor [24]. In addition, bacterial cells can reduce the number of potentially infectious bacteriophage particles by producing membrane vesicles [25]. If phage adsorption is successful, the host cell can subsequently prevent injection of phage NA by modification of the inner membrane proteins [26]. The infected bacterium can also identify the injected phage NA and degrade it via a restriction–modification system [27] or a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins system (CRISPR-Cas) [28]. Another possibility is preventing the replication of phage NA by secondary metabolites produced by the bacterial cell [29], or preventing the assembly of virions by inhibiting phage terminase or by scattering phage coat proteins [30]. If these bacterial cell defense mechanisms are not successful, the host cell may sacrifice itself for the benefit of others, resulting in cell death, and thus limiting the production of phage particles that may infect other bacterial cells in the environment (Figure 2) [31].
Microorganisms 10 01365 g002 550
Figure 2. Mechanisms of bacterial resistance to phage infection. Prevention of phage adsorption to the surface of bacterial cells due to alteration of the surface receptor or production of protective surface polysaccharides (1). Prevention of phage NA injection by modification of inner membrane proteins (2). Reducing the number of free infectious bacteriophage particles by producing membrane vesicles (3). Degradation of phage NA by the restriction modification system (4) or the CRISP-Cas system (5). Prevention of phage NA replication by secondary bacterial metabolites (6). Inhibition of the assembly of phage particles by blocking terminase or scattering envelope proteins (7). Limitation of phage particle production by induction of cell death (8).
However, bacterial strains rarely have more than one defensive mechanism against phage infection [32]. Alternatively, phages may possess mechanisms that help overcome the defensive machinery of the host cell. These include, for example, the ability to produce glycosidases to degrade host capsules, and thus unmask receptors to initiate phage infection [33]. Another mechanism is the ability of phages to produce hypervariable receptor-binding proteins that allow binding to modified host receptors [18,34]. Phages can also overcome the CRISPR-Cas system by mutating or deleting target sites for this system or expressing proteins that interfere with CRISPR-Cas [35]. An important mechanism is the ability of phages to modify their NA to avoid the host restriction–modification system [36], or even the ability of phages to encode their own CRISPR-Cas system that interferes with host defensive mechanisms [37]. All of these mechanisms determine the specificity of phages against bacterial hosts [12].

3. Properties of Phage Therapy

3.1. Antibiotic versus Phage Therapy

Phage therapy differs from antibiotic therapy in many ways. As mentioned in the previous section, phages attack a much narrower range of bacterial strains than antibiotics. The administration of antibiotics, especially broad-spectrum antibiotics, can often have adverse effects on the balance of the human microbiota resulting in, for example, post-antibiotic diarrhea [38]. Unlike antibiotic treatment, phage therapy is much gentler on the physiological microbiota [13]. However, this narrow host specificity of therapeutic phages sometimes requires precise knowledge of the infectious agent, which presupposes cultivation of the infection site and verification of sensitivity to commercial phage preparations, or the identification of suitable phages for individualized patient treatment [15]. A wider range of potential infectious agents is covered by polyvalent phage cocktails containing several types of phages having extended host specificity [13,39].
A huge advantage of therapeutic phage preparations over antibiotics is the absence of toxic side effects of phages against mammalian cells. Another advantage of phages is their bactericidal effect, as the lytic cycle results in the destruction of the host cell. Phages kill their host bacterial cells in the last phase of their lytic replication cycle, when new phage particles are released from the bacterium. This also increases the number of phage particles directly at the site of infection, and thus increases the probability of infection by other bacteria present [39].
Since the mechanism of action of phages is different from that of antibiotics, antibiotic resistance mechanisms do not affect phage efficacy. Therefore, bacteriophages may be used in the treatment of infections caused by resistant or multidrug-resistant bacterial strains [12,40].
The great potential of bacteriophages has also been observed during biofilm removal, where their ability to enter the biofilm by gradual lysis of individual bacterial layers is manifested. Biofilm can also be removed by the action of phage depolymerases [41]. An interesting alternative is to use a combination of antibiotics with phages [39].
Similarly, like all microorganisms, phages are also able to elicit an immune response in the human body. Detection of phages can occur through pattern recognition receptors. Subsequently, the phagocytes are activated and begin to penetrate the site of infection, which can also contribute to the elimination of bacterial agents [42].
Disadvantages of phage therapy include the potential formation of neutralizing antibodies, which could reduce the effectiveness of phages during their longer administration or repeated phage therapy with the same phages. Prevention of such phage “disposal” can be achieved by improving dosing regimens or by exchanging the phages used in therapy [12]. Another possible risk is that phage preparations may contain endotoxins released from the cell wall of host bacteria; this can be completely eliminated by appropriate phage suspension technology and efficient purification methods [16]. However, the release of endotoxins can also occur during the lysis of bacterial cells by phages in the patient’s body, which can trigger a cascade of immune responses and subsequent adverse events upon systemic administration. However, similar reactions may also accompany antibiotic therapy [39]. Due to the fact that phage preparations are predominantly administered locally and, more rarely, systemically, the risk of such reactions is very low [2,3,43,44,45,46,47,48].
 

3.2. Phage Administration

Phage preparations may be administered topically, orally, by aerosol, in the form of suppositories, intravenously, intraperitoneally, intramuscularly, or subcutaneously. The simplest and most widely used method of administration in clinical practice is the local application of a phage preparation, due to which it is possible to achieve high concentrations of phages at the site of infection. A limitation of this procedure is the risk of leaching the phage preparation from the application site, which can be avoided by using phages incorporated into the gel carrier or in the emulsion. The advantage of oral use of phage preparations is the possibility of using higher doses of the preparation. The disadvantage is that the acidic environment of the stomach can reduce the number of active phages [12]. The solution may be the use of phages on an empty stomach after neutralization of the stomach content with sodium bicarbonate [49], or in the form of microcapsules in which the phages are protected by biopolymers resistant to gastric acid and intestinal juice [50]. Phages can reach areas that are poorly congested in the form of aerosol, but the disadvantage is that there are high losses in phage concentration due to the presence of mucus. Better penetration of phage preparations through the mucus layer can be ensured by using depolymerases. For intravenous administration, the rapid systemic diffusion of phage particles is an advantage, but there is a danger of their rapid removal by neutralizing antibodies. The solution may be the selection of low-immunogenic phages. In the case of intramuscular and subcutaneous administration of phage preparations, phages are delivered directly to the site of infection, but only low concentrations are achieved. This limitation may be resolved by increasing the number of doses [12]. A comparison of the properties of phage and antibiotic therapies is summarized in Table 1.
Table 1. Comparison of aspects of phage and antibiotic therapies [12,39].

4. Phage Therapy at Present

Despite the fact that bacteriophages were discovered as early as 1917, the use of phage therapy was suppressed globally by the discovery and development of antibiotics. Phage therapy has a rich tradition in some countries of Eastern and Central Europe, especially in Georgia, Russia, and Poland. The Eliava Institute in Tbilisi [51] and Microgen in Moscow [52] are best known for their production of bacteriophage preparations. These institutions focus on the production of various commercial phage cocktails such as Bakteriofag-Stafilokokovyj, Sextaphag® (both NPO Microgen), Staphylococcal Bacteriophage, Fersisi Bacteriophage, SES-Bacteriophage, Intesti-Bacteriophage, Enko-Bacteriophage, and Pyo-bacteriophage (Eliava BioPreparations). Generally, they are recommended for local usage in patients with pyogenic infections, such as infections of skin and soft tissues, infections of the respiratory tract, infections of the eye, urinary tract infections, or decolonization of infectious agent carriers in the nasal cavity. Intesti-Bacteriophage and Enko-Bacteriophage are focused mainly on the treatment of gastrointestinal infections [53,54,55,56,57,58,59,60].
The Institute of Immunology and Experimental Therapy in Wroclaw [61] deals with phage therapy to a significant extent. In the Czech and Slovak republic, a STAFAL® phage preparation having an antistaphylococcal effect is registered and indicated for local use [62]. However, phage therapy is still not widely used in most countries due to the lack of efficacy benchmarks and well-established information on safety, approved manufacturing practices, and standard protocols for the treatment [12,63]. In connection with the development of bacterial resistance to antibiotics, efforts to produce safe and effective phage preparations have also been made in the West [64].
 

4.1. Phage Laboratory Studies

Despite the limited use of phage preparations, many have already been investigated in several in vitro and in vivo studies [65,66,67,68,69,70].
The effect of the antistaphylococcal preparation STAFAL® was demonstrated in the work of Dvořáčková et al., with significant efficacy in methicillin-resistant Staphylococcus aureus (MRSA) strains [40]. The high efficacy of Staphylococcal Bacteriophage and Pyo-bacteriophage was also demonstrated in the experimental study against MRSA strains in biofilm [65]. In vitro susceptibility of this bacterium was also proven in the study published by Verstappen; however, ex vivo and in vivo susceptibility was not established [66]. In contrast, phage treatment of mastitis in mouse models was successful [67].
The susceptibility of extended-spectrum beta-lactamase-producing Escherichia coli strains to phage preparations, such as SES-Bacteriophage, Intesti-Bacteriophage, Enko-Bacteriophage or Pyo-bacteriophage, was determined by Gundogdu et al. SES-bacteriophage was effective against 59.2% of the tested strains, and all other cocktails were active against more than 80% of the strains [68]. A more recent study described newly isolated phages that work well in resistant uropathogenic E. coli strains [69].
Namonyo et al. and Camens et al. discovered novel phages that are active against strains of Pseudomonas aeruginosa. Further analysis of the isolated PA4 phage revealed its good stability under various temperature and pH conditions, and the absence of gene-encoding antibiotic resistance mechanisms and the production of toxins [70,71]. In the study published by Fong et al., phages were able to reduce the biofilm formed by P. aeruginosa in vitro [72].
Phage efficacy against different mycobacterial species was demonstrated by several laboratory studies (reviewed by [73]).
 

4.2. Phage Case Studies

Several clinical cases in which phage therapy has been administered have also been published [2,3,44,45,46,47,48,74,75,76]. Jennes et al., described the case of a man in his 60s who developed a pressure ulcer during hospitalization that was colonized by a resistant strain of P. aeruginosa. Subsequently, the patient developed sepsis and was treated with colistin as the only effective antibiotic. The patient’s condition was complicated by acute renal failure, which led to the need to discontinue colistin. Phages whose efficacy had been demonstrated in vitro were used in experimental therapy. Phage therapy was administered intravenously to the patient. The patient’s kidney function improved within a few days and the collected blood cultures were negative. However, pressure ulcers remained colonized by strains of P. aeruginosa and other species of pathogenic microorganisms. Four months later, the patient developed a septic condition due to the presence of a strain of Klebsiella pneumoniae in the patient’s blood, leading to cardiac arrest and the death of the patient [2].
In another case, a 15-year-old patient with cystic fibrosis and various comorbid conditions underwent a lung transplant and was affected by disseminated Mycobacterium abscessus infection. For the treatment, a phage preparation was designed and applied intravenously; this was well tolerated, lung and liver function improved, and the skin nodes healed after 6 months. Phage-neutralizing antibodies were not detected [45]. Contradictorily, in the case of 81-year-old man infected by M. abscessus, intravenous phage treatment failed due to the phage-neutralizing antibody response [46].
In a 7-year-old cystic fibrosis patient infected with P. aeruginosa and S. aureus strains, there was no significant improvement in clinical condition after antibiotic therapy. Therefore, phage therapy was initiated using Pyo-bacteriophage, which was applied by nebulization. Treatment was administered nine times at intervals of 4–6 weeks and the quantity of the strain of P. aeruginosa was significantly reduced, but the preparation of the phage was not effective against the strain of S. aureus. Subsequently, bacteriophage Sb-1 was added to Pyo-bacteriophage to eliminate the S. aureus strain. The modified phage preparation was administered to the patient five times. The result was a significant reduction in the amount of S. aureus strain, with no serious side effects during treatment [74].
Phage therapy has also been used successfully in the treatment of patients with prosthetic joint infection. A 62-year-old diabetic with a history of complete knee arthroplasty overcame several episodes of prosthetic knee infection. Despite numerous surgeries and long-term antibiotic treatment, he was at risk of limb amputation due to persistent periimplantitis of the knee caused by K. pneumoniae. However, phage therapy was initiated, administered intravenously in 40 doses. In addition, he continued to receive minocycline due to a previous infection caused by strains of Enterococcus faecalis and Staphylococcus devriesei/hemolyticus, whose eradication was unsuccessful. This therapy led to remission of local symptoms, signs of infection, and restoration of knee function. The patient had no treatment-related adverse events and remained healthy for 34 weeks after cessation of treatment while still receiving minocycline [75].
In a double-blind, randomized study, a six-phage cocktail from Biocontrol Limited (BiophagePA) or placebo (glycerol-PBS solution) was applied to the ear canal in 24 patients with otitis media caused by P. aeruginosa. Treatment success was monitored after 7, 21, and 43 days, and revealed a statistically significant improvement in clinical status in patients treated with the phage compared to the control. No adverse reactions were reported in the phage group [3].
A 47-year-old patient after head trauma developed an abscess and ventriculitis caused by a multidrug-resistant strain of Acinetobacter baumanii. Intravenous phage therapy was initiated and, after 8 days, the patient’s cerebrospinal fluid was culture-negative for A. baumanii; however, it was positive for K. pneumoniae and S. aureus, and the patient died [47].
A very promising application of phages is their use in the treatment of urinary tract infections, due to the occurrence of resistant strains, limited possibilities of antibiotic treatment, and frequent occurrence of relapses in patients. In a study described by Ujmajuridze et al., nine patients who underwent urinary tract surgery and subsequently had a urinary tract infection received Pyo-bacteriophage phage preparation through a suprapubic catheter. The efficacy of this phage preparation was tested in vitro and adapted for the treatment of urinary tract infections (active against S. aureus, E. coli, Streptococcus spp., P. aeruginosa, and Proteus mirabilis). Treatment was administered twice a day for 7 days. In most patients, the pathogen titers decreased significantly and, in addition, no adverse events were reported during treatment [76].
A 60-year-old man with a left ventricular assist device was infected by multidrug-resistant P. aeruginosa. Phage preparation containing three phages was applied intravenously four times. The culture became negative and the patient’s condition improved [47].
Within the Phagoburn project [64], Jault et al. tested a phage preparation in the treatment of burns infected with P. aeruginosa. However, the efficacy of the preparation was lower compared to that of standard therapy (1% sufadiazine cream emulsion), probably due to insufficient phage titers at the site of infection [44].
Schooley et al. described a 68-year-old man with a pancreatic pseudocyst infected by A. baumanii. After unsuccessful antibiotic therapy, phage therapy was administered intravenously, with a marked improvement at 48 h. After 11 weeks of therapy, complete recovery was achieved [48].
 
 

5. Conclusions

In the post-antibiotic era, interest in phage therapy is increasing because lytic phages are able to destroy bacterial cells, and thus treat infections. Throughout evolution, bacteria have competed for survival by producing defensive mechanisms, and phages have developed methods to overcome these mechanisms. This review describes the advantages and disadvantages of phage therapy, various laboratory studies, and clinical cases where phage therapy has been administered successfully. The importance of phage therapy has been demonstrated, especially in the treatment of infections caused by multidrug-resistant bacterial strains. Therefore, phages can play an important complementary role to antibiotics.

Author Contributions

Conceptualization, M.S. and A.L.; Visualization, M.S.; Writing—original draft, M.S. and M.D.; Writing—review and editing, A.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by project PROMEGA_13/2022 and by Ministry of Education of Slovakia grant KEGA no. 002UK-4/2022.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Microbiota of Female Genital Tract – Functional Overview of Microbial Flora From Vagina to Uterine Tubes and Placenta

Review

Adriana Liptáková 1, Katarína Čurová 2, Jozef Záhumenský 3, Kristína Visnyaiová 3, Ivan Varga 4

1 Institute of Microbiology, Faculty of Medicine, Comenius University in Bratislava, Slovak Republic
2 Department of Medical and Clinical Microbiology, Faculty of Medicine, University of P. J. Šafárik in Košice, Slovak Republic
3 Department of Gynecology and Obstetrics, Faculty of Medicine, Comenius University in Bratislava and University Hospital Bratislava, Slovak Republic
4 Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava,

Physiological Research 71 (Suppl. 1): S21-S33, 2022
https://doi.org/10.33549/physiolres.934960

Summary
Microorganisms and eukaryotic human cells coexist in synergistic relationships in nearly every niche of the human body. The female genital tract consisting of the vagina, uterus with its cervix and endometrium, uterine tubes and ovaries – harbors its own typical microbiota, which accounts for 9 % of the total bacterial population in females. To this organ system, we also assigned the microbiome of the placenta, which has not been studied much until now. Among the spectrum of microbial species, the female genital tract is mainly dominated by Lactobacillus species, which are considered to be one of the simplest yet most important microbial communities. However, this relationship between macro- and micro-organisms seems to have a number of physiological functions, e.g., the vaginal and cervical microbiota have unique impact on reproductive health. The aim of this review was to provide current view on female genital tract microbiota and its role in reproductive health. We describe in detail the association of vaginal or tubal epithelium with microbiota or the role of microbiota in normal placental function.
 
Key words
Microbiota; Female genital tract; Reproductive functions; Lactobacillus spp.

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Introduction

Microorganisms and eukaryotic human cells coexist in synergistically in nearly every niche of the human body. The microbiota in the human body play a critical role in maintaining human wellbeing and are associated with the pathogenesis of various diseases [1]. These communities of microorganisms can be found in the skin, respiratory tract, alimentary tract, and other tissue sites, each with their own functional capabilities [2]. The goal of the International Human Microbiome Project, which began in 2007, was to analyze the genomic information of microorganisms in healthy adults. Samples from the nose, mouth, skin, intestines and vagina were taken from healthy adult volunteers. Microbes were identified based on the sequence of 16S ribosomal ribonucleic acid target regions, and gene content information was obtained by whole genome sequencing. Analyses have shown species variations as well as gene composition in individuals as well as within different sites on the human body. For example, the bacteria colonizing the intestinal tract are different from bacteria that colonize the oral cavity, skin, or other sites. The intestine is the locality with the most significant taxonomic and genetic diversity, and the vagina seems to be the least complex. 

Each microenvironment (e.g., intestine, skin surfaces, and vagina) has its own unique microbiome. Most people have so-called basic microbiome, which is 95 % identical for all individuals. The basic microbiome helps perform normal metabolic functions, stimulates innate immunity, prevents the colonization of unwanted pathogens [2,3]. In addition, an analysis of the microbiota within the female reproductive tract revealed a microbiota continuum along the whole tract, which is indicative of a non-sterile environment [4]. Female genital tract comprising the vagina, uterus (especial interest is focused on the cervix and endometrium), uterine tubes and ovaries. It harbors its own typical microbiota (Fig. 1), which accounts for 9 % of the total bacterial population in females [5,6].

Fig. 1. An overview of the composition of a microbiota in the reproductive tract of healthy reproductive age woman

Among the spectra of microbial communities, the female genital tract microbiota, mainly dominated by Lactobacillus species, are considered to be one of the simplest yet most important microbial communities, and the cervicovaginal microbiota have vast impact on the reproductive health of females [7]. For example, lactobacilli help regulate the pH value of the vagina to inhibit the growth of other bacteria and to prevent undesirable microbial colonization and infection through their adhesion to the vaginal epithelial cells [8]. However, the composition, diversity, and dynamics of the microbiota within the uterine cavity of reproductive-aged women have not been fully unveiled, and as the uterine cavity seems to be an essential part of the female genital tract. More efforts are needed to further illustrate the interaction between microbial communities in the vagina and uterus. Current evidence have shown that female genital tract microbial communities are closely associated with gynecological disease. However, the impact of microbial communities in the uterine cavity on female fertility and the underlying mechanism still remain largely unclear [9,10].

Overview of methodical approaches in microbiota research

To investigate the composition of microbiota in the genital tract, samples are collected from individual locations (organs) throughout the genital tract of healthy reproductive-aged females. For vaginal microbiota analyses, most groups so far performed vaginal swabs and vaginal secretions sampled by swabs. Some groups take urine or vaginal aspirates with sterile catheter. Two types of samples are collected in order to analyze cervical microbiota, swabs of cervical mucus and endocervical swabs. Collection of these samples is noninvasive process, but in case of cervix samples the risk of contamination with vaginal species is higher [6]. The most common samples used in the detection of endometrial microbiota are endometrial swabs, endometrial biopsies and endometrial fluid sampled by catheter. These specimens are taken by invasively during laparoscopy or laparotomy from minimally invasive surgery, providing samples or through the cervical canal. The problem of transcervical samples might be their contamination by the vaginal microbiota. The specificity of sampling route was tested to determine the distribution of bacteria. The results of this research yielded high similarity of bacterial distribution in the transcervical samples to that in samples taken by opening the uterus during surgery [4]. To determine uterine tubes microbiota, samples are obtained by laparoscopic access, specifically by salpingectomy, by direct biopsies of the distal portion of the uterine tube during laparoscopic procedures. Currently, hysteroscopy is considered an ideal diagnostic procedure for assessing the vaginal walls, cervical canal, uterine cavity, endometrium, and tubal ostia [11].

Traditionally, to investigate bacterial or fungal diversity, culture-dependent techniques were used. The bacterial composition of the vaginal microbiota was detected by microscopy after Gram staining of vaginal smear. Some standardized criteria based on Nugent scoring and Amsel’s criteria were used to analyze the presence of Gram-positive Lactobacillus, compared to Gram-negative and Gram-variable bacteria such as Gardnerella, Atopobium and Mobiluncus. The cultivation process allowed these microbes to be identified [12, 13]. Culture-based techniques have various disadvantages. Principally, culture conditions are unsuitable for all microbial species, which leads to many species being undetected. If microbial species have been isolated, they are unable to recognize and culture-based procedures are not adjustable to high-throughput investigation. The fact is that 1 % of bacteria survive and form colonies on agar plates. Special platforms based on DNA/RNA and protein analysis have been established to recognize the real diversity of the microbiota [14]. Most of our current knowledge on the genital microbiota is based on qualitative and semiquantitative descriptive research using cultivation-dependent methods.

The introduction of culture-independent molecular-based methods provided new information about the composition of  genital   flora and abnormal colonization, which has supplemented existing knowledge from culture-dependent techniques [13]. The identification of microbiota is based on genomic DNA analysis using these techniques. The DNA is isolated from specimen by a variety of next-generation sequencing tools. These procedures can also be used to isolate individual microbial species from mixed cultures. Following DNA isolation, two methods may be used to examine microbiota. The first is whole metagenome sequencing that focuses entirely on the sequencing of microbial DNA present in the sample. The second is marker gene sequencing that targets sequencing of a particular locus in all genomes. In this way the 16S rRNA gene is the most commonly used marker gene specific to archaea and bacteria, the 18S rRNA gene and 28S rRNA gene are specific for eukaryotic organisms and the internal transcribed spacer is specific for fungi. The complete procedure includes microbial DNA extraction, PCR amplification of the selected gene and next-generation sequencing. By such methods that do not rely on microbial cultivation, it is possible to determine the composition of the microbiota in the female genital tract at the species level much accurate compared with the culturebased techniques [6,13,15,16].

Healthy vaginal microbiota

The vaginal microbiota is a dynamic microbial ecosystem that regularly undergoes fluctuations throughout a female’s life and throughout the menstrual cycle [17]. A mutual relationship exists between woman reproductive physiology and vaginal microbiota. Over the last decades, female reproductive health has been at the forefront of research as strong evidence supports bidirectional relationships between the vaginal and cervical microenvironments and cervical cancer, viral acquisition and persistence, gynecologic and obstetric diseases and other benign conditions like cervical ectopy [18]. Physiological hormonal changes that start from birth and continue till post-menopause, may affect the vaginal microbiota. On the other hand, vaginal microbiota can also affect reproductive physiology [19]. The vaginal microbiota differs among individuals due to variations in sexual activity, vaginal douching / intravaginal washing, regional disparity and other factors [4,20]. A previous study has investigated the microbial compositions in three sites of the vagina, including the introitus, midpoint, and posterior fornix, and concluded that there was little variation in species across the three sampling sites, with Lactobacillus species being dominant in all sites [2].

From the past, the vaginal microbiota of healthy females in their reproductive age is defined as Lactobacillus dominated microflora, producing sufficient quantity of lactic acid with pH values ˂ 4.5 [21]. The composition of the vaginal microbiota has critical implications for the susceptibility to sexually transmitted infections, miscarriage, and spontaneous preterm delivery [22-28]. The mechanism is in part attributed to the action of Lactobacillus spp., many of which provide broadspectrum protection via antimicrobial substances, mainly lactic acid, narrow-ranging bacteriocins and wide-ranging hydrogen peroxide. These products are suggested to play various important roles in host defense [29-31].

The composition and structure of the vaginal microbiota have been described adequately using conventional techniques (microscopy, cultivation) and non-conventional techniques, especially sequencing. Although often dominated by lactobacilli, the vaginal microbiota is also frequently composed of a collection of facultative and obligate anaerobes. Based on the abundance and composition of vaginal bacterial species in reproductive age females detected by molecular based techniques, five major microbial community state types (CSTs) are established. These CSTs are differentiated by dominant species and pH values. CST-I, CST-II, CST-III and CST-V are characterized by abundance of Lactobacillus crispatus, L. gasseri, L. inners and L. jensenii, respectively. CST-IV harbors higher ratios of strictly anaerobic bacterial genera Prevotella, Atopobium, Dialister, Gardnerella and Sneathia and low levels of genus Lactobacillus. Depending on the Lactobacillus abundance, average pH values range from 4.0 ± 0.3 (CST-I) to 5.3 ± 0.6 (CST-IV) [21, 32]. Two sub-states exist in CST-IV. The CST-IV-A is characterized by species of genera Anaerococcus, Peptoniphilus, Corynebacterium, Prevotella, Finegoldia and Streptococcus. The CST-IV-B contains Atopobium, Gardnerella, Sneathia, Mobiluncus, Megasphera and other taxa of order Clostridiales [31, 32]. Among these five states, the CST-I, II, III and V exist in 89.7 % white women, 80.2 % Asian women, 61.9 % Afroamerican women and 59.6 % Hispanic women. The CST-IV state predominates in healthy Afroamerican and Hispanic women (40 %), but also represents the most common dysbiosis state (bacterial vaginosis) [32]. The vagina of child has CST-IV of vaginal microbiota and neutral pH value. Some reproductive aged women show switching between CSTs over a short span, other remain consistent. These conversions are elicited by menstruation [33, 34]. Post-menopause leads to shift of vaginal microbiota from lactobacilli to microbial diversity (CST-IV) and rise in pH [35]. Additionally, a higher number of lactobacilli are found in pregnant when compared to non-pregnant females [36]. The above research results indicate that the vagina harbors a huge microecosystem containing billions of microbes. Data from 110 reproductive aged women revealed that the vagina contains 1010 – 1011 bacteria [4].

The relationship between intravaginal douching and vaginal health is interesting, whereas nearly half of American women reported washing the inside of the vagina during the past month (based on a sample of sexually active women living in Los Angeles) [37]. According to some studies, intravaginal washing is associated with decreased vaginal colonization with beneficial lactobacilli [38]. The use of commercial vaginal douching products containing vinegar, iodine or baking soda is likely not be beneficial for vaginal health. Hesham et al. [39] demonstrate in vitro that vaginal douching products increase vaginal epithelial cell death and secretion of pro-inflammatory cytokines, suggesting the potential for epithelial disruption. After exposure to vinegar-based douche, Lactobacillus crispatus and L. jensenii – two classic beneficial lactobacilli – induce greater production of the pro-inflammatory cytokine IL6. In vivo, analyses of vaginal fluid cytokine levels demonstrates higher levels of pro-inflammatory cytokines in women who use douching products [40]. Based on these findings, vaginal douching could potentially increase the risk of all genitourinary infections, including urinary tract infections, and support clinical recommendations to avoid douching [39].

In addition to bacteria, the vaginal microbiota of healthy reproductive aged women also contains fungi that form vaginal mycobiota. Using culture-dependent techniques researchers isolated vaginal fungi in approximately 20 % of the asymptomatic women. Predominant species Candida albicans (72-91 %) was followed by non-albicans Candida species (C. glabrata, C. tropicalis, C. parapsilosis) [41]. Studies based on sequencing techniques yielded Candida species in significantly higher frequency, which accounted for 64.5 % of the participants involved. The predominant part of this mycobiota was C. albicans (82 %), followed by C. dubliniensis, C. parapsilosis, C. krusei, Candida sp. V104616 [42]. Studies based on co-culturing of vaginal yeast and bacteria suggested that bacteria inhibit Candida yeast to hyphae switch, maintain low numbers of Candida in vagina and compete with yeast cells for adhesion sites on epithelial receptors owing to its higher affinity.

Lactobacillus abundance and low Candida number along with their interactions play an important role in maintaining microbiota balance [43, 44].

Overlap of urogenital and vaginal microbiota

Recent studies applying culture-independent methods have allowed for the detection of a quantifiable and diverse urinary microbiota [45-49], and these findings have been validated with quantitative enhanced culture methods [50,51]. Several organisms commonly found in the vagina have been observed in urine samples [47,52]. Bacterial strains isolated from the urinary bladder and vagina have been found to be functionally and phylogenetically similar [53]. In one study, voided urine samples demonstrated more similarity to paired vaginal swabs than to paired supra-pubic needle aspirates or transurethral catheterized samples [54]. This suggests that the microbiota of some types of urine samples may more closely resemble vaginal microbiota than other urine sample types; however, there is also similarity at the genus level between paired vaginal and trans-urethral catheterized samples [55]. Given the overlap between the genitourinary and vaginal microbiota, it was hypothesized that voided urine may be used as a proxy for vaginal community assessment in research studies utilizing 16S rRNA gene amplicon sequencing. To evaluate the use of urine as a proxy for vaginal swabs, Brown et al. [56] compared the microbiota of paired mid-vaginal swabs with the microbiota of urine samples collected using “cleancatch” or “random-catch” methods from reproductiveaged females, and paired mid-vaginal swabs and randomcatch urine samples from peri/post-menopausal women. The first void of the initial urine stream is collected for random-catch urine samples, while for urine collected via the clean-catch method, the labia are cleaned with an antibacterial wipe and mid-stream urine is collected. While the microbiota of both random-catch and clean-catch urine samples might be similar in composition to vaginal microbiota because of shared species, RC urine may contain a higher proportion of vulvovaginal bacteria due to contamination from the urine stream washing over the labia, resulting in a better proxy of the vaginal microbiota than clean-catch urine. To our knowledge, clean-catch and random-catch urine samples have not been assessed in conjunction with the vaginal microbiota and, although the concordance between the urinary and vaginal microbiota of peri/post-menopausal women has been studied [55], they have not been evaluated separately from reproductive-age women. Peri/post-menopausal women have different vaginal [57, 58] and urinary [59] microbiota compared to reproductive-age women, and may carry lower bacterial loads [60]. These differences may affect the extent to which the genitourinary and vaginal microbiota overlap.

Interaction between vaginal epithelium and vaginal microbiota

More than 50 different species of bacteria may live in a woman’s vagina, with lactobacilli being the predominant microorganism found in healthy adult females [61]. Some studies, however, describe more than 200 bacterial species of normal and the abnormal vaginal microbiota influenced by genes, ethnic background and environmental and behavioral factors [62]. Lactobacilli are relevant as a barrier to infection and are important in the impairment of colonization by pathogens, owing to competitive adherence to adhesion sites in the vaginal epithelium and their capacity to produce antimicrobial compounds. Due this, women without vaginal Lactobacillus strains may be susceptible to nonindigenous and potentially harmful microorganisms [61]. The human vaginal microbiota is a critical determinant of vaginal health. These communities live in close association with the vaginal epithelium and rely on host tissues for resources [63].

The human vaginal surface lined by stratified squamous epithelium has a large surface area (mean 87.46 cm2, measured by vinyl polysiloxane casting) and is the first mucosal surface contacted by sexually transmitted pathogens [64]. The stratified vaginal epithelium undergoes differentiation and contains several distinct layers: the deepest and mitotically active basal layer, the parabasal layer, the intermediate layer where the glycogen amount increase, and the superficial layer, where cells continuously die. During the regulated cell death of the vaginal epithelial cells, glycogen is released on the surface of the epithelium. This glycogen is metabolized and their product – lactic acid – forms acidic microenvironment of vagina [65]. However, the interaction between lactobacilli and other microorganisms and the vaginal epithelium and the entire vaginal microenvironment is much more complex than it might seem at first glance. The superficial layers of the vaginal epithelium provide a unique microenvironment that maintains vaginal health by fostering endogenous lactobacilli and retaining critical mediators of acquired and innate immunity, as also leukocytes may also penetrate and traverse the superficial layer following placement in the vaginal lumen [66]. It seems that the vaginal epithelial cells actively regulate membrane adhesiveness to co-ordinate bacterial adhesion. From biochemical point of view, bacterial adhesion forces were dramatically decreased by depleting the epithelial cell membrane of cholesterol or sub-membrane cortical actin [67].

Key nutrients for lactobacilli include sugars produced when glycogen is degraded. But most genital isolates of lactobacilli are not able to use glycogen as an energy source in vitro. An important role during glycogen degradation plays α-amylase enzyme, which has been demonstrated in vaginal fluid. Until now, it is unclear whether α-amylases are produced solely by the vaginal epithelium, bacteria in the vagina, or both [68, 69].

The understanding of the interaction between normal vaginal microflora and vaginal epithelium is crucial for new methods of bacterial vaginosis treatment. Vaginosis is defined as a condition experienced by most women at least once in their lifetime. This condition arises due to the imbalance in the microbiome of the vaginal ecosystem. Most of the pathogens of this disease are organisms which are commonly found in a normal healthy vagina. Recent studies have provided insights into the relationship between the vaginal microbiome environment and bacterial vaginosis symptoms. In the Lactobacillusdominated vaginal microbiome, various antimicrobial substances are produced, including lactic acid, bacteriocins, and hydrogen peroxide, which play essential roles in protecting against potential pathogens [70]. The standard of care treatment for bacterial vaginosis is antibiotics. Recently, vaginal microbiome transplant and vaginosis treatment with probiotics, efforts to restore the normalcy in the vaginal environment, are becoming new and popular treatments [70, 71].

Cervical microbiota

The microbiota correlation among vaginal, cervical and endometrial samples within an individual is very strong. These findings have been shown by Wee et al. [72] who has analyzed cervical mucus samples of healthy women and determined dominance of genus Lactobacillus (L. crispatus, L. inners), accounted for 97.56 %. In this study quantitative PCR method was used.  In another study cervical mucus samples were analyzed by sequencing and shotgun sequencing and dominance of Lactobacillus was confirmed. Several studies exhibited higher diversity of bacterial genera in endocervical samples. In one study prevalence of Lactobacillus and Prevotella was reported [73]. Another study published Lactobacillus was dominant genus, followed by Gardnerella, Veilonella, Prevotella, Sneathia, Fusobacterium [74]. Winters et al. [75] reported dominance of Acinetobacter (49 %), followed by Pseudomonas, Cloacibacterium and Lactobacillus. Chen et al. [4] analyzed endocervical samples of healthy women and women with different conditions by 16S rRNA sequencing. Lactobacillus was the most recovered genus (almost 75 %), followed by Gardnerella, Streptococcus, Atopobium, Prevotella and Pseudomonas. Some studies underline the link among high diversity of species within cervical microbiota and different gynecological issues.

Persistent high-risk human papillomavirus infection (HPV) is undoubtedly the main carcinogen leading to cervical intraepithelial neoplasia and cervical cancer. However, studies have found that not all patients with cervical HPV infection experience development into cancer. According to recent studies of Wang et al. [76] and Lin et al. [77], the relationship between cervical microbial diversity or dysbiosis may be related to the severity of high-risk HPV infection and cervical intraepithelial neoplasia as a precancerous condition.

Endometrial microbiota

Until recently, the endometrium was considered a sterile environment. Several studies have now reported that the endometrium harbors a functional microbiome in physiological conditions. Detection of bacteria through culture-independent techniques of endometrial samples have confirmed a unique microbiota harboring 100 to 1000 times less bacterial amount than that of the vagina. This low biomass microbiota is characterized by high diversity. Like the normal vaginal microbiota, the endometrium of healthy asymptomatic women is often colonized by lactobacilli that represent dominant group of bacteria [78-80].

The configuration of healthy bacterial microbiota is not clearly defined as some studies suggest Lactobacillus to be dominant and representative genus of healthy endometrium and some other studies point to other bacterial genera [6]. Franasiak et al. [81] examined endometrial samples and detected Lactobacillus and Flavobacterium the most abundant genera. Analysis of endometrial fluid samples of fertile women revealed the highest incidence of Lactobacillus (71.1 %), followed by Gardnerella, Bifidobacterium, Streptococcus and Prevotella [60]. Garcia-Velasco et al. [82] classified 2 types of endometrial microbiota, Lactobacillusdominated with ˃90 % of Lactobacillus spp. and nonLactobacillus-dominated with ˂90 % of Lactobacillus spp. plus ˃10 % of other bacteria. However, non-Lactobacillusdominated microbiota have been identified in the genital tract of healthy and asymptomatic women, suggesting that in the absence of pathological signs, this microbiota could be considered normal [80].

The similarity between bacterial genera in vaginal and endometrial samples of healthy women supports the hypothesis that uterine cavity is colonized mainly by vaginal bacteria coming from the vagina by ascending route. Specific physiochemical or biological conditions in the uterus of some women may lead to colonization by the bacterial community that differs significantly from the vaginal community. In such cases, collection and analysis of endometrial samples is important for diagnostics of the microbial state of the uterus [80].

Uterine tubes and ovarian microbiota

Both uterine tubes and ovaries display highly variable microbial communities among women. In contrast with the acidity of vaginal pH value, these organs harbor a variety of bacteria growing in mildly alkaline conditions. Lactobacillus sp. is present in lower ratio than in vagina or cervix [4].

Pelzer et al. [83] have demonstrated using both culture-dependent and culture-independent techniques, that in the absence of infection, the human uterine tube is not a sterile site. Uterine tubes in asymptomatic women contain diverse microbial communities, which are affected by hormones and antibiotics, and display biogeographical tropism. Microbiota is represented by members of the phylum Firmicutes, most notably Staphylococcus sp., Enterococcus sp., and Lactobacillus sp.  Other highly abundant and prevalent taxa include pseudomonads (Pseudomonas sp. and Burkholderia sp.) and known genital tract anaerobes Propionibacterium sp. and Prevotella sp. The results of microbial profiling are consistent with cultivation for most cohorts, with Staphylococcus sp. dominating both the culture-dependent and culture-independent results. Community profiles differ significantly between the left and the right uterine tubes. Lactobacillus sp., Enterococcus sp. and Prevotella sp. are more abundant within the left tube versus the right tube, whilst Staphylococcus sp. is more abundant within the right tube. The microbial community within the ampulla demonstrates a significantly greater abundance of Enterococcus sp. and P. acnes when compared to the isthmus [83]. For the sake of the complexity of knowledge, we will add that in contrast with the vaginal epithelium, the simple columnar epithelium of the uterine tubes contains numerous specific cells of the immune system. As our previous research shows, these immunologically active cells are mostly regulatory T- lymphocytes [84]. The occurrence of these intraepithelial T- lymphocytes together with secretion of tubal fluid containing cytokines and chemokines by tubal epithelial cells [85] cause pathogen inhibition inside uterine tube. Probably because of both mentioned factors, the presence of lymphatic follicles in the wall of uterine tubes, as the sign of chronic inflammation, is rare and is present during histological examination only in 2.1 % of surgically removed uterine tubes [86].

Miles et al. [73] detected Bacteroides, Corynebacterium, Lactobacillus, Coproccocus and Hymenobacter in uterine tubes, and Lactobacillus, Corynebacterium, Escherichia, Blaudia in ovarian samples surgically removed during total hysterectomy and bilateral salpingo-oopherectomy. Further analyses of ovarian follicular fluid by Pelzer et al. [87] revealed L. iners, Actinomyces spp., Corynebacterium auromuosum, Fusobacterium sp., Prevotella, or Staphylococcus sp. being colonisation more prevalent in the left than in the right ovary. Based on this finding we can suppose that the follicular fluid is not sterile. Microorganisms colonizing follicular fluid and the ensuing cytokine response could be a further as yet unrecognized cause and/or predictor of adverse assisted reproduction techniques outcomes and infertility.

Zhou et al. [88] analyzed the diversity and composition of the microbiota from 25 ovarian cancer tissues and 25 normal distal uterine tube tissues by 16S rRNA sequencing. Results of sequencing showed that the diversity and richness indexes were significantly decreased in ovarian cancer tissues compared to tissues from normal distal uterine tubes. The ratio of Proteobacteria and Firmicutes was notably increased in ovarian cancer, which revealed that microbial composition change might be associated with the process of ovarian cancer development. The authors assume that the microbial composition change may be involved in the initiation and progression of ovarian cancer via influencing and regulating the local immune microenvironment of uterine tubes except for regular pathways [88].

Placental microbiota

The placenta exhibits its own unique microbiome, with a low abundance but a metabolically rich microbiome. The combination of 16S rDNA and wholegenome shotgun metagenomic techniques indicate that the placental microbiota largely consists of nonpathogenic commensal microbiota from the phyla of Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and Fusobacteria. By such composition, the placental microbiota mostly resembles the microbiota of the oral cavity and the deep endometrium of non-pregnant women rather that of the adjacent vaginal microbiota [2, 89]. Since fetal viability, growth and development are completely dependent on optimal placental function, the recent finding of a placental microbiome in healthy pregnancy may implicate a role for the bacteria in normal fetal growth and development [90].

Conclusion

Results   of            culture-dependent               and         cultureindependent studies have revealed composition, diversity and functions of healthy female genital tract microbiota

References

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Conflict of Interest

There is no conflict of interest.

Acknowledgements

This work was supported by The Slovak Research and Development Agency Grant No. APVV-18-0499, by the Ministry of Health of the Slovak Republic under the grant number 2019/34-UPJŠ-6 and by Ministry of Education of

Slovak Republic under the grant number KEGA 002UK4/2022.

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