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In vitro study evaluating the effect of selected oral probiotic supplements on Trichomonas vaginalis.


Dominika Kowalczyk



Introduction

Probiotics are defined as live microorganisms that, when administered in an adequate amount, exert a beneficial effect on the human body [1]. Their beneficial effects and effectiveness must be documented in studies, which is why fermented foods cannot be included outright in the group of probiotics. Among the microorganisms with probiotic properties, the majority are lactic acid bacteria (LAB).

The probiotic group includes microorganisms of the genus Lactobacillus, Bifidobacterium and, among others, Enterococcus. Among the bacteria of the genus Bifidobacterium, strains that are investigated probiotics include: B. lactis, B. bifidum, B. breve, B. adolescentis, B. infantis, B. longum [2, 3]. There are also other types of bacteria that are studied as probiotics, for example: Bacillus cereus var. Toyoi, Escherichia coli Nissle, Propionibacterium freudenreichii. Fungi such as unicellular yeasts of the genus Saccharomyces cerevisae and Saccharomyces boulardii are also considered microorganisms with probiotic applications.

Trichomonas vaginalis is the most common sexually transmitted protozoan that is pathogenic to humans. It is characterized by an oval, round or spindle-shaped trophozoite, the presence of five flagella and axostyle. The preferred location of the parasite is most often the vagina, less commonly the perivaginal glands and the urogenital tract in women and men, as well as the prostate. It causes a disease called trichomoniasis, which is nowadays very often asymptomatic, which is dangerous in itself, since infection with this protozoan is one of the factors which may promote infection with HIV.

If symptoms are present, in women they are characterized by, among others, the presence of a foamy greenish or yellowish vaginal discharge, often with an unpleasant smell, vulvar pruritus, sometimes with vaginal pain and burning. The woman may also experience pain during vaginal intercourse. In men, trichomoniasis is often asymptomatic, and if it manifests, it's via urethritis, burning sensation during urination, and swelling and redness of the glans and foreskin.

Diagnostics is based on tests detecting Trichomonas vaginalis and confirming the presence of this motile protozoan in the sample collected from vagina (direct observation). The sensitivity of this method oscillates around 65%, however, it increases when the microscopic examination is performed immediately after collecting the material or after culturing it for 24-48 h in Diamond's medium. Immunofluorescence methods and nucleic acid amplification tests (NAAT), which are often the gold standard in highly developed countries, are characterized by very high sensitivity, however, they are more complex and require more time and financial means. Primary regimen for treating trichomoniasis is based on the administration of metronidazole or tinidazole to both the sick person and the infected sexual partner [4, 5].


Materials and Methods

The study was carried out in the microbiological and parasitological laboratory of the Department of Microbiology, Jagiellonian University Medical College in Cracow, Poland. In the study, four oral preparations with gynaecological probiotics were used. Their final target and place of activity was the vagina. Preparations were purchased from funds granted by the Dean of the Faculty of Medicine of the Jagiellonian University Medical College in response to the submitted application. The purchased probiotic preparations included:

• Trivagin – 3 billion of four LAB strains:
-Lactobacillus rhamnosus (0.9 billion),
-Lactobacillus gasseri (0.9 billion),
-Lactobacillus fermentum (0.6 billion),
-Lactobacillus plantarum (0.6 billion).

• Iladian – 6 billion of three LAB strains:
-Lactobacillus rhamnosus (2.5 billion),
-Lactobacillus reuteri (2.5 billion),
-Lactobacillus gasseri (1 billion).

• LaciBios Femina – 5 billion of two LAB strains:
-Lactobacillus rhamnosus GR-1 (2.5 billion),
-Lactobacillus reuteri RC-14 (2.5 billion).

• prOVag – 1 billion of three LAB strains:
-Lactobacillus gasseri 57C (0.5 billion),
-Lactobacillus fermentum 57A (0.25 billion),
-Lactobacillus plantarum 57B (0.25 billion).

Two strains of T. vaginalis were used in the study:
- KAT strain from the collection of the Department of Microbiology of the Jagiellonian University Medical College and
- ATCC PRA-92 reference strain from the USA.

Bacteria. One capsule of each probiotic was filled into four tubes with de Man, Rogosa, Sharpe (MRS) medium and left for 24 hours in anaerobic culture at 37°C (Figures 1-4). After this time, dilutions of probiotic preparations were made to show 105 colony forming units (CFU)/ml, 107 CFU/ml, 109 CFU/ml. Then the study was continued in a parasitological laboratory. Thirty tubes were prepared, containing 5 ml of Diamond's medium, 0.5 ml of bovine serum and two drops of antibiotic (not inhibiting the bacteria). Three drops of 109 CFU/ml probiotic were added successively to four test tubes: to the first Trivagin, to the second Iladian, to the third LaciBios and to the fourth Provag, with a control of the probiotic preparations.

figure1
Figure 1. Gram-stained specimen from the Iladian preparation culture.
[please click on the image to enlarge]


figure2
Figure 2. Gram-stained specimen from the Trivagin preparation culture.
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figure3
Figure 3. Gram-stained specimen from the prOVag preparation culture.
[please click on the image to enlarge]


figure4
Figure 4. Gram-stained specimen from the LaciBios Femina preparation culture.
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Protozoan. Trichomonas vaginalis strains were added to the remaining twenty-six tubes. One half contained the strain from the Department (hereinafter referred to as KAT), while the other contained the reference strain ATCC PRA-92. One tube from each strain was set aside as a T. vaginalis control. Three drops of probiotic preparations at the two remaining concentrations were added to the remaining twenty-four tubes. Cultivation was carried out for 24 and 48 hours at 37°C. All perfomed in triplicate.

Results

The Tables 1-4 show the experiment results. Table legends are presented at the end of tables.

Table 1. Results of the experiment with Trichomonas vaginalis KAT strain after 24h culture.
table1
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Table 2. Results of the experiment with Trichomonas vaginalis ATCC PRA-92 after 24h culture.
table2
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Table 3. Results of the experiment with Trichomonas vaginalis KAT strain after 48h culture.
table1
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Table 4. Results of the experiment with Trichomonas vaginalis ATCC PRA-92 after 48h culture.
table1
[please click on the image to enlarge]




Legend to Tables 1-4:
"–": all trophozoites are dead/visual field
"single trophs": most trophozoites are dead
"+": few moving trophozoites/visual field
"++": many moving trophozoites/visual field
"+++": very numerous trophozoites/visual field


Considering the average results from all experiments on both Trichomonas vaginalis strains, it can be concluded that the most potent antagonistic in vitro activity against T. vaginalis was shown by the probiotic preparation Trivagin, followed (in decreasing order) by: Iladian, prOVag, LaciBios Femina.

Discussion

The use of probiotics in various gynaecological ailments has its justification in the literature. Gynaecological probiotics are used to support antibiotic therapy in bacterial vaginosis, vulvovaginal candidiasis and other infections. Their use is also justified when maintaining normal vaginal microflora and thus preventing the occurrence of intimate infections. Bacteria of the genus Lactobacillus are the most commonly used microorganisms in the production of gynaecological probiotic preparations, as the vaginal environment is mainly inhabited by lactic acid bacteria [6].

Conclusions

Our experiments have shown that the probiotic supplements intended for oral consumption, with the target site of action in the vagina, inhibit to a different extent, the Trichomonas vaginalis parasite in laboratory conditions in vitro.

References

[1] Hill C et al. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 2014; 11:506-14.
[2] Holzapfel WH, Haberer P, Geisen R, Schillinger U. Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr 2001; 73:365S.
[3] Kleerebezem M, Vaughan EE. Probiotic and Gut Lactobacilli and Bifidobacteria: Molecular Approaches to Study Diversity and Activity. Ann Rev Microbiol 2009; 63:269-90.
[4] Kochan P, Pietrzyk A. Atlas tematyczny: Trichomonas vaginalis. KOHASSO, Kraków 2017.
[5] CDC - DPDx - Trichomoniasis. Access valid on February 12, 2020: https://www.cdc.gov/dpdx/trichomoniasis/index.htm.
[6] Cribby S, Taylor M, Reid G. Vaginal Microbiota and the Use of Probiotics. Interdiscip Perspect Infect Dis 2008; 2008:1-9.

Conflict of interest: PK was the thesis supervisor of the author.

Acknowledgements: This article is based on the Master Thesis by the author bearing the same title: "Badanie in vitro oceniające wpływ wybranych doustnych suplementów probiotycznych na rzęsistka pochwowego".

Author’s affiliations (at the time of study performance):
1 Dietetics studies, Faculty of Medicine, Jagiellonian University Medical College, Cracow, Poland.
2 Department of Bacteriology, Microbial Ecology and Parasitology, Jagiellonian University Medical College, Cracow, Poland.

Corresponding author:
Dominika Kowalczyk
ul. Czysta 18
31-121 Kraków
Tel. +48 515 571 421
e-mail: dominika.kowalczyk1994@gmail.com

To cite this article: Kowalczyk D. In vitro study evaluating the effect of selected oral probiotic supplements on Trichomonas vaginalis. World J Med Images Videos Cases 2020; 6:e19-24.

Submitted for publication: 16 February 2020
Accepted for publication: 20 May 2020
Published on: 31 May 2020













































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