INTRODUCTION:

The healthy vaginal microbiota mostly consists of different Lactobacillus strains which maintain optimal microbial balance¹. The qualitative and quantitative composition of vaginal microorganisms can be changed due to physiological processes in women’ organisms and affecting general factors². The dysbiosis vaginal conditions occur according to various unfavorable effects. The most widespread condition is bacterial vaginosis from which suffer up to 50 % of reproductive age women population^{3, 4}. It is important that nearly half of patients with bacterial vaginosis have asymptomatic course of the disease. Women with bacterial vaginosis complain of itching of vagina’s mucous membrane, vaginal discharge usually of white-yellowish color and unpleasant «fishy» odor ⁵. Also, there is an increasing of pH because of microorganisms’ disbalance³. The second popular vaginal disorder is vulvovaginal candidiasis which affect more than 3/4 of women of childbearing age^5,6,7,8,9,10.Near half of patients with vulvovaginal candidiasis have one episode of pathology recurrence and 5% suffer from recurrent vulvovaginal candidiasis that occurs 4 and more times during the year⁷. Both bacterial vaginosis and vulvovaginal candidiasis impair quality of life and negative influence on intima health^4,6.

The common treatment of bacterial vaginosis and vulvovaginal candidiasis includes administration of antibiotics and antifungal medicines, but the level of recurrence amounts up to 50%^1,8,11. So, it is important to use drugs that can increase this rate or prevent pathologies. Probiotics have these properties and are known as «live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host» according to the definition of the Food and Agriculture Organization^9,12. One of the most often group of bacteria which consist in compositions of drugs with probiotic activity is Lactobacillus¹³. According to the literature medicines containing Lactobacillus can renew and keep the vaginal microbiota on optimal level or destroy biofilms associated with bacterial vaginosis like antibiotics, but can be administered during long period without the risk of antibiotic resistance causing^14,15,16.

The basic principles of Lactobacillus action are co-aggregation with pathogens, adhesion toepithelial cells, auto-aggregation, production of antimicrobial substances like bacteriocins, biosurfactants, hydrogen peroxide and decreasing of the pH level due to synthesis of organic acids especially lactic acid by glucose fermentation^17,18.

The ability of Lactobacillus to adhere to vagina epithelia is the one type of barrier production mechanism for the prevention of pathogens colonization and probiotic activity^19,20. Adherence to mucous epithelia is regulated by bacterial cell wall mediators like sortase-dependent proteins, lectin molecules, exopolysaccharides, lipoteichoic acid and S-layer proteins^21,22.

Antagonistic activity is depended on the synthesis of different metabolites like flavonoids, glycosides compounds, steroids, indole, indazole, benzoic acid, gluco-phospholipids, salicylic acid, ferulic acid, caffeic acid, lactic acid, acetic acid, formic acid, linoleic acid, phenyllactic acid, vanillic acid, azelaic acid, hydrocoumaric acid, hydroferulic acid, hydrocaffeic acid, 2,3-butadione, reuterin, acetaldehyde, hydrogen peroxide, hydroxyl radical, peptides such as the bacteriocins and their derivatives²³.

Lactobacilli are resistant to the range of antibiotics due to their biological properties, but development of pharmaceutical industry and spread of resistance to antibiotics lead to evaluation of probiotic strain sensitivity to antibiotics^24,25.

We developed a composition of pessaries with probiotic activity containing Lactobacillus casei IMV B-7280 strain, synthesized in Zabolotny Institute of Microbiology and Virology, NAS of Ukraine²⁶.

The aim of this article is to study properties of pessaries with probiotic activity containing L. casei IMV B-7280 strain, such as lactic acid production, antagonistic properties against pathogens, adhesion to epithelial cells and sensitivity to antibiotics and anti-fungal medicines.

MATERIALS AND METHODS:

The objects of this study were experimental samples of pessaries with probiotic activity containing L. casei IMV B-7280, prepared by molding in laboratory conditions.

The lactic acid production was determined by acid-base titration with 0.1 M sodium hydroxide using as indicator 1 % phenolphthalein solution. The experimental samples of pessaries were placed in test tubes with Man-Rogosa-Sharpe (MRS) medium and put on the water bath with temperature 37±1^оС. The content of test tubes was homogenized and incubated with the same temperature in thermostat during 48 hours. After that the lipophilic phase of suppository base was removed by the sterile pipet and suspension for titration was prepared by mixing and diluting with purified water. Titration is finished after changing on light pink color of solution with potentiometrically pH control. The index of lactic acid production (^oT) was determined by the formula:

°T = A * K * V, where

A – the volume of the sodium hydroxide solution, mL;

K – correction factor by the sodium hydroxide solution;

V – the volume of the suspension for titration, mL.

The adhesion to epithelial cells was determined by Brylis method using the buccal epithelial culture. The medial adhesion index, epitheliocytes coefficient and the microorganism index of adhesiveness were determined for the characteristic of L. casei IMV B-7280 adhesive properties^27,28. The last index was calculated as a ratio of medial adhesion index and epitheliocyte coefficient and multiplicated on 100. The adhesivity was evaluated as very low with average adhesion index from 0 to 1.00; low – from 1.01 to 2.00; medium – from 2.01 to 4.00; high – more than 4.01. The microorganism strain was evaluated as non-adherent with the microorganism index of adhesiveness lower than 1.75; low-adherent with the microorganism index of adhesiveness from 1.76 to 2.5; medial-adherent – from 2.51 to 4.0 and high-adherent – more than 4.0.

The antagonistic activity of pessaries containing L. casei IMV B-7280 strain was determined by the method of perpendicular strokes. Staphylococcus aureus 8325-4, S. aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922 (F-50), Proteus vulgaris ATCC 4636, Bacillus subtilis ATCC 6633, Candida albicans ATCC 885-653 were used as test-microorganisms. The experimental samples of pessaries were placed in sterile test tubes with isotonic sodium chloride (37±1^oC). Then test tubes were placed on water bath and their content was melted with the temperature 38±1^оС for the liquid condition. After melting the test tubes content were mixed and homogenized, the lipophilic phase of suppository base was removed by the sterile pipet. The obtained suspension was inoculated in Petri dishes in MRSA medium with two parallel strokes on the whole diameter. After 24hours test microorganisms were plated in perpendicular way to the microorganism growth. The antagonistic activity was evaluated by the calculation of the size of the zone of no growth of the test cultures. The antagonistic activity was considered as low with the size of the zone of no growth from 0 to 10mm, medial – from 10 to 20 mm and high – more than 20mm.

The sensitivity of pessaries containing L. casei IMV B-7280 to antibiotics and anti-fungal medicines was determined by agar disk-diffusion method ^{29, 30}. The experimental samples of pessaries were placed in sterile test tubes with isotonic sodium chloride (37 ± 1 ^oC). Then test tubes were placed on water bath and their content was melted at a temperature 38 ± 1 ^оС for the liquid condition. After melting the test tubes content were mixed and homogenized, the lipophilic phase of suppository base was removed by the sterile pipet. The obtained suspension was inoculated in Petri dishes with agar medium, after the disks with antibiotics and antimycotics were placed on the surface. The cultivation conducted during 24hours in thermostat with the temperature 37±1^оС.

The size of the zone of no growth of L. casei IMV B-7280 strain up 10.9 mm is considered as resistant, 11.0 – 15.9mm – moderately sensitive, more than 16mm – sensitive.

RESULTS:

After research of lactic acid production, we obtained the mean of °T = 183.8 ± 1.5. This result shows that pessaries with L. casei IMV B-7280 have the ability to lactic acid production and increasing pH in vagina.

Next stage of our research was experiment of adhesion properties of L. casei IMV B-7280 strain in the pessaries composition (Table 1).

Table 1: Results of adhesion properties research of L. casei IMV B-7280 in the composition of pessaries with probiotic activity

Indicator	Value	Note
Medial adhesion index, units	6.79 ± 0.016	high (More than 4.01)
Epitheliocytes coefficient, %	87.49 ± 0.03	–
The microorganism index of adhesiveness, units	7.76 ± 0.02	high adhesive – (More than 4.0)

* n = 5, M ± SD

Medial adhesion index– is the number of microorganisms that have attached to the surface of a single cell of the used culture, in all fields of view.

Epitheliocytes coefficient – is a ratio of the number of cells in the used culture that have lactobacilli adhered to their surfaces, to the total number of cells in the culture, expressed as a percentage.

The microorganism index of adhesiveness – the average number of bacteria that exhibit adhesive properties is considered. Obtained results demonstrate that L. casei IMV B-7280 strain in the composition of pessaries with probiotic activity is high adhesive and can provide high adhesion level during intravaginal administration of drug in pessaries dosage form.

The results of L. casei IMV B-7280 strain antagonistic activity in the composition of pessaries are presented in Table 2.

Table 2: Results of antagonistic activity of L. casei IMV B-7280 in the composition of pessaries with probiotic activity research

Test-strain	The zone of growth inhibition of test-strain, mm	Note
Staphylococcus aureus 8325-4	11.3 ± 0.23	++
Staphylococcus aureus ATCC 6538	8.8 ± 0.17	+
Pseudomonas aeruginosa ATCC 9027	17.4 ± 0.11	++
Escherichia coli ATCC 25922 (F-50)	13.6 ± 0.15	++
Proteus vulgaris ATCC 4636	13.8 ± 0.13	++
Bacillus subtilis ATCC 6633	10.1 ± 0.13	++
Candida albicans ATC 885-653	10.8 ± 0.17	++

* n = 5, M ± SD

+ – low activity

++ – medial activity

+++ – high activity

The results of research of L. casеi IMV B-7280 antagonistic properties showed medial antagonistic activity against pathogenic and opportunistic microorganisms. The highest level of antagonism – 17.4 ±0.11 mm was detected against test strain P. aeruginosa ATCC 9027, the lowest level – 8.8±0.17 mm against S. aureus ATCC 6538.

Final stage of our research was the determination of L. casei IMV B-7280 strain in the composition of pessaries sensitivity to antibacterial and antifungal substances^31,32: oxacillin, ampicillin, piperacillin, amoxicillin, amoxicillin/clavulanic acid, cefazolin, ceftriaxone, cefoperazone, imipenem, lincomycin, clindamycin, azithromycin, ciprofloxacin, pefloxacin, vancomycin, levofloxacin, furazolidone, linezolid, nystatin, metronidazole, econazole, miconazole, clotrimazole, and ketoconazole. The results are presented in Table 3.

Table 3: Sensitivity of L. casei IMV B-7280 in the composition of pessaries with probiotic activity to antibiotics and anti-fungal medicines

The name of medicine, ATC-group	The diameter of the zone of growth inhibition, mm	Note
Penicillins, beta-lactam antibiotics (J01C):
Oxacillin	6.12 ± 0.08	R
Ampicillin	21.7 ± 0.08	S
Piperacillin	19.8 ± 0.08	S
Amoxicillin	23.1 ± 0.17	S
Amoxicillin/ clavulanic acid	23.4 ± 0.23	S
Other beta-lactam antibiotics (J01D):
Cephalosporins 1^st generation:
Cefazolin	13.3 ± 0.13	MS
Cephalosporins 3^rdgeneration:
Ceftriaxone	12.5 ± 0.28	MS
Cefoperazone	20.3 ± 0.15	S
Carbapenems:
Imepenem	17.5 ± 0.23	S
Macrolides, lincosamides (J01F):
Lincomycin	22.2 ± 0.1	S
Clindamycin	19.8 ± 0.11	S
Azithromycin	24.8 ± 0.2	S
Fluoroquinolones (J01MA):
Ciprofloxacin	12.6 ± 0.23	MS
Pefloxacin	17.5 ± 0.22	S
Levofloxacin	12.0 ± 0.23	MS
Other antimicrobial and antiseptic agents (G01A X):
Furazolidone	5.5 ± 0.18	R
Glycopeptide antibiotics (J01ХА):
Vancomycin	4.7 ± 0.18	R
Other antibacterial agents (J01X):
Linezolid	6.2 ± 0.11	R
Nystatin	3.6 ± 0.13	R
Imidazole derivatives (G01AF):
Metronidazole	6.4 ± 0.08	R
Econazole	7.5 ± 0.22	R
Miconazole	5.1 ± 0.17	R
Clotrimazole	4.6 ± 0.21	R
Ketoconazole	3.9 ± 0.16	R

* n = 5, M ± SD

R – resistant

MS – moderately sensitive

S – sensitive

It was established that L. casei IMV B-7280 strain in the composition of pessaries with probiotic activity is resistant to glycopeptide antibiotic vancomycin, oxacillin from the penicillin group, as well as linezolid, nystatit, furazolidone, and imidazole derivatives used as antifungal drugs^33,34.

The largest zones of growth retardation are observed in relation to penicillins belonging to beta-lactam antibiotics (except oxacillin), macrolides and lincosamides, as well as cefoperazone, pefloxacin and imepenem from the group of carbapenems, which indicates the sensitivity of L. casei IMV B-7280 strain to these antibacterial drugs. The tested strain shows moderate sensitivity to cephalosporins (cefazolin and ceftriaxone), fluoroquinolones (ciprofloxacin and levofloxacin).

DISCUSSION:

For pessaries as dosage form the determination of main quality control indicators such as organoleptical properties, average mass, disintegration, pH, microbiological purity, identification, quality control etc. is traditional³⁵. But for pessaries with probiotic activity, it is important to evaluate the specificity of active pharmaceutical ingredient. Thus, research of biological properties of L. casei IMV B-7280 strain as active ingredient it the composition is very important. Results of quality control, biological properties research, specific pharmacological activity, toxicological properties indicate the effectiveness and safety of tested drug.

The most important characteristic of probiotic bacteria is providing colonization resistance, which means protection against the invasion of both bacteria and toxins/toxic products of various origins into the internal environment of the body. In the mechanism of colonization resistance, the antagonistic activity of probiotics plays a significant role. This refers to the ability to colonize the mucous membrane, consisting of microorganisms adhering to epithelial cells, competing for binding receptors, and blocking the adhesion and colonization of the mucous membrane by pathogenic and opportunistic microorganisms. This process involves humoral and cellular defense factors of the host organism.

The main requirements for probiotic microorganism strains are as follows: the strains should be isolated from healthy individuals and identified by species using phenotypic and genotypic characteristics; they should have a genetic passport; possess natural resistance to antibacterial agents; exhibit colonization potential; demonstrate a wide spectrum of antagonistic activity against pathogenic and opportunistic microorganisms; not suppress the activity of normal microflora; be safe for humans, including immunological safety; production strains should maintain stability in biological activity and meet technological demands.

According to the general theory, a probiotic should possess two criteria for selection as an effective strain in the treatment of urogenital tract infections: the ability to colonize the host without any adverse side effects and the capability to inhibit urogenital pathogens, thereby preventing the consequences associated with antibiotic intake and recurrence prevention.

A strain of lactobacilli should possess the following characteristics: viability, the ability to grow (at a specific pH value), production of lactic acid and bacteriocins, and the capacity for colonization.

Our results of biological properties research are consistent with literature data and investigations of other authors. For example, E. M. Babych et al. researched biological properties of Lactobacillus strains during different cultivation conditions and highlighted that for relevant results it is very important to be sure that quantity of probiotic strain in the one dose of medication not less than should be. Because this is directly influence on the effectiveness of drug. So, the quantity content it is obligatory part of the specification on probiotic medicine in each dosage form.

L. Singhal et al. studied the sensitivity of 29 Lactobacillus strains and researched that all strains were sensitive to nitrofurantoin and resistant to norfloxacin, gentamicin, and cotrimoxazole excluding L. paracasei.

S. Saduakhasova et al. researched ability of Lactobacillus strains to be active pharmaceutical ingredients in drugs composition and showed that the probiotic consortia had high antagonistic, adhesive properties, and resistance to metronidazole.

V. Ocana et al. determined that vaginal probiotic lactobacilli don’t display uniform sensitivity to antibiotics, but resistance to metronidazole suggests that lactobacilli could be simultaneously used in the bacterial vaginosis treatment.

S. Coudeyras et al. suggested that the probiotic strain L. rhamnosus can be as a protective agent against both bacterial vaginosis and candidiasis since it was able to adhere to vaginal cells and to antagonist the growth of vaginosis-associated pathogens.

P. Hutt et al. study revealed that the production of different antimicrobial metabolites is highly strain specific and that the metabolites are not correlated with each other; the best potential probiotic candidate could be found among L. crispatus strains.

So, the value of lactic acid production, antagonistic activity, adhesion properties and sensitivity to antimicrobials and antifungals depends on the type of Lactobacillus strain. But not all of them can be used as potential active pharmaceutical ingredients in medicine formulations.

CONCLUSIONS:

The results of our study of lactic acid production ability of the strain L. casеi IMV B-7280 in the composition of pessaries show the high ability of this strain to produce lactic acid and reduce the pH value in the vagina. The study of the adhesive properties of the strain L. casеi IMV B-7280 in the composition of pessaries demonstrates that this strain is highly adhesive, thus allowing for a high degree of adhesion when intravaginally administering the drug in the form of pessaries. The results of the study on the antagonistic activity of the strain L. casеi IMV B-7280 in the composition of pessaries indicate moderate antagonistic activity against pathogenic and opportunistic microorganisms, which is permissible for use in the treatment and prevention of bacterial vaginosis and vulvovaginal candidiasis. The study of the sensitivity of the strain L. casеi IMV B-7280 in the composition of pessaries to antibacterial and antifungal medicines demonstrates that this drug can be used in complex treatment of vaginal dysbiosis with certain antibiotics and antifungal drugs, which is effective for achieving a therapeutic effect faster and preventing recurrences.

Therefore, the developed drug can be used in the complex treatment of vaginal dysbiosis disorders together with certain drugs, which makes it possible to improve therapy and contribute to achieving a faster and higher-quality clinical effect.

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Received on 03.12.2024 Revised on 24.04.2025

Accepted on 27.06.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3767-3772.

DOI: 10.52711/0974-360X.2025.00542

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