vaginal dysbiosis and its correction with vaginal probiotic neoprobio taras lyaskovsky phd,...

Vaginal dysbiosis and its correction with vaginal probiotic Neoprobio

Taras LyaskovskyPhD, Laboratory of Lactic Acid Bacteria

Zabolotny Institute of Microbiology and Virology National Academy of Sciences, Kiev, Ukraine

email: [email protected]

XXXII Kongres Polskiego Towarzystwa GinekologicznegoŁodz, September 3, 2015

dr Taras Lyaskovsky | Zabolotny Institute of Microbiology and Virology NAS | Ukraine

Zabolotny Institute of Microbiology and Virology of National

Academy of Sciences of Ukraine


• Institute is the main scientific organization in Ukraine in the field of microbiology and virology research.

• 17 departments and labs• State depositary of non-pathogenic microorganism • Accredited laboratory in the sphere of control of

biological products in Ukraine• Publish “Microbiological Journal”




• Department of Physiology of Industrial Microorganisms• Department of antibiotics• Department of General and soil microbiology• Department of Biochemistry microorganisms• Department of phytopathogenic bacteria• Department of Biology extremal microorganisms• Department of Physiology and Systematics of micromycetes• Department of Genetics of microorganisms• Department of phytopathogenic viruses• Department of microbiological processes on solid surfaces• Department mycoplasmology• Department of Molecular Genetics of Bacteriophages• Division problems interferon and immunomodulators• Department of Innovation and Technology Transfer• Laboratory of secondary metabolites micromycetes• Laboratory reproduction of viruses• Laboratory biological polymer compounds



Bacterial Vaginosis is the most common vaginal infection in women ages 15-44.

http://www.cdc.gov/std/bv/stats.htm


Bacterial Vaginosis is the most common cause of vaginal symptoms among women.

The prevalence in USA is estimated to be 21.2 million (29.2%) among women ages 14–49.

Most women found to have BV (84%) reported no symptoms.

Women who have not had sex can still be affected by BV (18.8%), as can pregnant women (25%), and women who have ever been pregnant (31.7%).

Prevalence of BV increases based on lifetime number of sexual partners.

Source: Koumans EH, Sternberg M, Bruce C, McQuillan G, Kendrick J, Sutton M, Markowitz LE. The prevalence of bacterial vaginosis in the United States, 2001-2004; associations with symptoms, sexual behaviors, and reproductive health. Sex Transm Dis. 2007 Nov;34(11):864-9.


BV is a polymicrobial clinical syndrome resulting from replacement of the normal hydrogen peroxide producing Lactobacillus sp. in the vagina with high concentrations of anaerobic bacteria (e.g., Prevotella sp. and Mobiluncus sp.), G. vaginalis, Ureaplasma, Mycoplasma, and numerous fastidious or uncultivated anaerobes.


What is Bacterial Vaginosis?

Factors promoting Bacterial Vaginosis

• uncotrollable drugs administration – mainly antibiotic • frequent use of contraceptive products • changes of hormonal status at menstrual cycle• stress• travelling• suppression of immune system• inflammation processes• other


Composition of the vaginal microbiota in women of reproductive age

Genetic analysis with the use of 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays for:

• healthy women• intermediate• Bacterial Vaginosis


Lactobacillus iners

Lactobacillus crispatus

Other Lactobacillus species

Gardnerella vaginalis

Atopobium vaginae

Eggerthella

Prevotella

BVAB 1

BVAB 2

Finegoldia magna

Megasphaera type 1

Megasphaera type 2

Sneathia sanguegens

Leptotrichia amnionii

BVAB-NV7

Mobiluncus curtisii/mulieris

Other non-Lactobacillus species

0 5 10 15 20 25 30 35 40 45 50

Microorganism %Lactobacillus iners 38.1Lactobacillus crispatus 43.2Other Lactobacillus species 5.4Gardnerella vaginalis 7.2Atopobium vaginae 0.6Eggerthella 0.001Prevotella 0.2BVAB 1 0.02BVAB 2 0.01Finegoldia magna 0.1Megasphaera type 1 0.2Megasphaera type 2 0Sneathia sanguegens 0.1Leptotrichia amnionii 0.1BVAB-NV7 0.002Mobiluncus curtisii/mulieris 0.002Other non-Lactobacillus species 4.8

Healthy women



Intermediate

Microorganism %Lactobacillus iners 43.9Lactobacillus crispatus 1.2Other Lactobacillus species 6.1Gardnerella vaginalis 30.5Atopobium vaginae 1.5Eggerthella 0.2Prevotella 0.9BVAB 1 0BVAB 2 0.5Finegoldia magna 0.1Megasphaera type 1 4.2Megasphaera type 2 0.001Sneathia sanguegens 0.2Leptotrichia amnionii 2.4BVAB-NV7 0Mobiluncus curtisii/mulieris 0.01Other non-Lactobacillus species 8.3Lactobacillus iners

Lactobacillus crispatus


Gardnerella vaginalis

Atopobium vaginae

Eggerthella

Prevotella

BVAB 1

BVAB 2

Finegoldia magna

Megasphaera type 1

Megasphaera type 2

Sneathia sanguegens

Leptotrichia amnionii

BVAB-NV7

Mobiluncus curtisii/mulieris


0 5 10 15 20 25 30 35 40 45 50

Bacterial Vaginosis (BV)

Microorganism %Lactobacillus iners 6.6Lactobacillus crispatus 0.3Other Lactobacillus species 0.3Gardnerella vaginalis 29.1Atopobium vaginae 7.1Eggerthella 1.2Prevotella 13.2BVAB 1 6.1BVAB 2 6.3Finegoldia magna 0.1Megasphaera type 1 9.8Megasphaera type 2 0.1Sneathia sanguegens 4.3Leptotrichia amnionii 6.8BVAB-NV7 0.8Mobiluncus curtisii/mulieris 0.2Other non-Lactobacillus species 7.9

Lactobacillus iners


Atopobium vaginae

Prevotella

BVAB 2

Megasphaera type 1

Sneathia sanguegens

BVAB-NV7


0 5 10 15 20 25 30 35



Report of a Joint FAO/WHO Working Group onDrafting Guidelines for the Evaluation

of Probiotics in Food London Ontario, CanadaApril 30 and May 1, 2002

Food and Agriculture Organization of the United Nations & World Health Organization

Probiotic strain screening


• Strain identification– Strain identification by phenotype (API test systems) – Strain identification by genotype (sequencing)

• Functional characterization– In vitro tests– Animal studies

• Safety assessment – In vitro and/or animal– Phase 1 human study

• Clinical trials (Phase II (DBPC), Phase III (effectiveness in comparing with standard treatment)

• Deposit strain in IMV culture collection

EVALUATION OF PROBIOTICS


PROBIOTIC SCREENING STRAIN IDENTIFICATION

• Study of phenotype characteristics of LAB

• Study of genotype characteristics of LAB


Fermentation of 49 carbohydrate substrates (API 50 CH)

Colony: size, surface, colorCells: form, size, positioning,

Motion

Growth on different media (substrates)

6,5% NaCl; pH 9,6; 40% bileGrowth at temperatures 150С,

300С, 370С, 450С, 500С

Gas production from glucose, NH3 from arginine

Higrolitic activity: gelatin, eskuline

Acidification and the growth in milk

PHENOTYPE CHARACTERISTICS


Gel picture showing the PCR amplified product of probiotic strains of lactic acid bacteria :

М- marker, 1- E.durans Sm, 2-E.faecium 77Д, 3-Str.thermophilus S8, 4- L.plantarum 200 Д

The result of the sequence of amplification products of DNA fragment of the strain L. plantarum 200 Д with primers Lp3_sp and Lp2 ( 543 b.p. fregment)

[GGC TGC TGG CAC GTA GTT AGC CGT GGC TTT CTG GTT AAA TAC CGT CAA TAC CTG ААС AGT TAC TCT CAG ATA TGT TCT TCT TTA АСА АСА GAG TTT TAC GAG CCG AAA CCC TTC TTC ACT CAC GCG GCG TTG CTC CAT CAG ACT TTC GTC CAT TGT GGA AGA TTC CCT ACT GCT GCC TCC CGT AGG AGT TTG GGC CGT GTC TCA GTC CCA ATG TGG CCG ATT ACC CTC TCA GGT CGG СТА CGT ATC ATT GCC ATG GTG AGC CGT TAC CCC ACC ATC TAG СТА ATA CGC CGC GGG ACC ATC CAA AAG TGA TAG CCG AAG CCA TCT TTC AAG CTC GGA CCA TGC GGT CCA AGT TGT TAC GCG GTA TTA GCA TCT GTT TCC AGG TGT TAT CCC CCG CTT CTG GGC AGG TTT CCC ACG TGT TAC TCA CCA GTT CGC CAC TCA CTC AAA TGT AAA TCA TGA TGC AAG CAC CAA TCA ATA CCA GAG TTC GTT CGA CTT GCA TGT ATT AGG CAC GCC GCC AGC GTT CGT CCT GAG CCA GGA TCA ААС TCT ]

99% identical to gene 16S рРНК of L. plantarum, presented in GenBank.

GENOTYPE CHARACTERISTICS

Electrophoregram of amplification products with the use of genus specific primers


PROBIOTIC STRAIN SCREENING FUNCTIONAL CHARACTERIZATION

• Adhesion Activity• Antagonistic Activity• Lysozyme Synthesis• Vitamin Synthesis

• Interferon Induction


ADHESION ACTIVITY

Adhesion activity is studied in vitro on the model of pig enterocytes


LACTIC ACID BACTERIA ADHESION TO EPITHELIUM OF DIFFERENT BIOTOPES OF HUMAN ORGANISM

BUCCAL VAGINAL INTESTINAL

x 4000


ANTAGONISTIC ACTIVITY

Pribiotic strains

of LAB

Bacillus cereus ATCC 11778

Staph. epidermitis ATCC 12228

Proteus vulgaris ATCC 6896

Staphylococcus aureus ATCC 12228

Klebsiella pneumonia ATCC 10031

Candida albicans ATCC 885-653

Pseudomonas aeruginosa ATCC

9027

Escherichia coli ATCC 25922


ANTAGONISTIC ACTIVITY SYNERGISM OF PROBIOTIC LAB

Bacillus cereus

Escherichia coli

Proteus vulgaris Staphylococcus aureus

Pseudomonas aeruginosa

Candida albicans

+0123456789

10

E.durance Sm

0123456789

10

L. plantarum 200D

Path

ogen

s gr

owth

inhi

bitio

n zo

nes,

mm



TEST (Growth inhibition by LAB ) CONTROL

Growth inhibition of gas producing putrefactive microflora by LAB probiotic strains

контроль (фіз.розчин)

L.plantarum 200Д Str.thermophilus (Sn+Sm)

суміш трьох штамів (1:1:1)

0

10

20

30

40

50

60

70

Num

ber o

f gas

pro

duci

ng p

utre

facti

ve

mic

roor

gani

sms,

%

Control Physiologic solution

Blend of strains

E.durance Sm


L.plantarum 200D

Growth inhibition of gas producing putrefactive microflora by LAB probiotic strains


LYSOZYME SYNTHESISLy

ses

of m

icro

cocc

us z

one

diam

eter

, m

m

Micrococcus lysodeikticus

lysozyme assay



VITAMIN SYNTHESIS

L. plantartum 3 L. plantartum 45 L. plantartum 200D

0

100

200

300

400

B5 (NICOTINAMIDE)0

20

40

60

80

100

120

B2 (RIBOFLAVIN)

0

0.2

0.4

0.6

0.8

1

H (BIOTIN)0

0.2

0.4

0.6

0.8

1

1.2

B6 (PYRIDOXINE)

μg/ml

0

1

2

3

4

5

B1 (THIAMINE)

μg/ml


INTERFERON INDUCTION BY LACTIC ACID BACTERIA IN EXPERIMENTS IN VITRO

CulturesInterferon activity INF AU/ml on media

№5 (bean broth) №7 (enzyme lysate cornmeal)

Streptococcus thermophilus Sn 0 320

E.durance Sm 1280 320

Str. thermophilus S24 640 1280

Str. thermophilus S8 160 640

E. faecium 77D 640 80

Lactobacillus plantarum 195D 0 0

L plantarum 200D 0 0

L. acidophilus 4356 0 0

Суміш: Sn +Sm + 200D 0 0

Полі {І}: полі {Ц} 2560 2560

Контроль середовища 0 0


OPTIMISED BLEND OF LAB

Bacterial vaginoses

Neoprobio

• Lactobacillus plantarum 200D

• Enterococcus faecium 77D

• Enterococcus durance Sm


• Interferon induction

Bacterial vaginoses

Neoprobio

• High adhesion activity (provide fast colonization of vaginal canal)

• High antagonistic activity (provide inhibition of growth of pathogenic microflora)

• Synthesis of biologically active substances (H2O2, lysozyme, vitamins

OPTIMISED BLEND OF LAB

UNIQUE COMPOSITION

Lactic acid bacteria blendLactoseAscorbic acidFolic acidHard fat



FUNCTION OF EACH INGREDIENT

LACTIC ACID BACTERIA (LAB)

BLENDLACTOSE ASCORBIC ACID FOLIC ACID HARD FAT

Probiotic properties of the

product

Starting and protective

ingredient for growth of lactic

acid bacteria

Provide acidic conditions which

create more favorable

condition for the growth of LAB then other BV

microflora

Provide acidic conditions and makes positive

effect on vaginal epithelium

regeneration.

Base of the suppository and protective media

for LAB. Slow melting of the

suppository provide

controlled release of LAB and effectively

colonies the vaginal canal by

LAB


CLINICAL TRIALS OF VAGINAL OVULES WITH PROBIOTIC LAB

BLEND


Name of the studyClinical studies of efficacy and tolerability of

XXX (vaginal suppositories with probiotic lactic acid bacteria) in treatment of patients with vaginal disbacteriosis (Bacterial Vaginosis).

CLINICAL TRIALS


Clinical base: Ministry of Health of Ukraine, Shupik National Medical Academy of post-graduate education Department of Obstetrics, Gynecology and Reproduction, Hospital No 4

Investigation Product:

XXX (vaginal suppositories with probiotic lactic acid bacteria)

Type of study: Under restricted programStudy design: Open, comparative, controlled, randomized studyStudy methods: Clinical study. General laboratory analysis, cytological, bacterioscopic,

bacteriological analysis of vagina discharge

Patients: Vaginal disbacteriosis in patients 18-40 years of ageNumber of patients:

Investigation group – 40 (Metronidazole+probiotic); Control group – 30 (Metronidazole);

Efficacy criteria: - clinical symptoms normalization dynamics;- dynamics of bacteriological, bacterioscopic, cytological analysis of vaginal discharge for disbacteriosis.

Objectives:

- study the efficacy of the study medication in vaginal disbacteriosis patients.- study the tolerability of the product and possible adverse product-related events. - compare the findings of treatment between the investigating and control groups.

CLINICAL TRIALS


Selection criteria for the patients in the trial

• Clinical symptoms (vaginal discharge that is usually thin and grayish white, vaginal odor (foul-smelling or unpleasant fishy odor), itching irritation.

• Microbiological examination (dominative microflora is Gardnerella vaginalis, Prevotella and other, Lactobacilli in low quantities).

• pH greater than 4.5.

CLINICAL TRIALS


Treatment

• Metronidazole 500 mg orally twice a day for 7 days

• Vaginal ovules with probiotic LAB blend twice a day for 10 days

CLINICAL TRIALS

Day 7 after start of the treatment

Microorganism

Investigation group (Combined treatment

(Metronidazole + vaginal ovules with probiotic LAB

blend)

Control group (Metronidazole)

% %

Lactobacillus iners 22 3.6

Lactobacillus crispatus 29.4 0.3Other Lactobacillus species 5.4 2.4

Gardnerella vaginalis 16.3 17.3

Atopobium vaginae 4.1 3.7

Prevotella 6.4 7


CLINICAL TRIALS


Day 20 after start of the treatment

Microorganism

Investigation group (Combined treatment

(Metronidazole + vaginal ovules with probiotic LAB

blend)

Control group (Metronidazole)

% %

Lactobacillus iners 25.4 7.9

Lactobacillus crispatus 36 3.5Other Lactobacillus species 7.2 8.4

Gardnerella vaginalis 14.3 16.4

Atopobium vaginae 5.1 3.9

Prevotella 5.8 6.2

CLINICAL TRIALS


Conclusions

Combined treatment (Metronidazole + vaginal suppositories with probiotic LAB blend) was effective in achieving therapeutic cure or in restoring healthy vaginal lactobacilli

• Fast colonization with beneficial microflora

• Decrease the risk of BV relapse

• Decrease the risk of local adverse events of standard treatment

• Treatment with Metronidazole reduced colonization with BV-associated bacteria, but was not effective in achieving therapeutic cure or in restoring healthy vaginal lactobacilli

CLINICAL TRIALS


PRODUCTION OF LAB BLEND

LAB blend is produced by Nutraceutix Inc, USA at a GMP certified facility.Guaranteed required quality parameters.


STRAINS INDENTIFICATION

As probiotic features of the lactic acid bacteria are strain dependent the strains for each batch of production are re-identified before and after the production to validate that the LAB blend conceits of the same probiotic strains. This identification is done by DNA sequencing technique.


MANUFACTURE OF FINAL PRODUCT

Neoprobio ovules are manufactured in Poland by FARMINA SP. Z O.O., KRAKÓW. A unique technology of low temperature production has been specially developed to allow safe processing of the LAB, keeping a therapeutic concentration of 109 to 107 of viable LAB cells per ovule. The facility is regularly audited according to EU GMP standards.


NEOPROBIO IS NOT ANOTHER VAGINAL PROBIOTIC!

NEOPROBIO IS UNIQUE!


UNIQUE OVULE FORM

Ovules provide controlled release of LAB and promote fast colonization of healthy microflora.

Capsules are not the correct vaginal form (may cause individual reactions to the compound of the capsule which contains unfavourable biological material and the rest of cultivation media).


UNIQUE OVULE FORM

Ovules provide direct therapeutic concentrations of LAB to vaginal canal.

The use of oral tablets or capsules decreases concentration of LAB which pass through the aggressive environment of the stomach and then depend on an unproven mechanism of „finding their way” from from anal canal into the vaginal canal.


LAB of the blend have been tested in vitro as well as in vivo and possess main characteristics:

• Interferon induction

• High adhesion activity (provide fast colonization of vaginal canal)

• High antagonistic activity (provide inhibition of growth of pathogenic microflora)

• Synthesis of biologically active substances (H2O2, lysozyme, vitamins

UNIQUE LAB BLEND


Unique composition helps create conditions for fast microflora balance change.

API of Neoprobio consists of tested and scientifically proved probiotic LAB according to WHO requirements. Validated technology of production and testing ensure that exactly specific probiotic strains are incorporated in the ovule.

UNIQUE LAB BLEND


• Neoprobio is indicated for the prevention and treatment of vaginal dysbiosis of different etiologies.

• Neoprobio is indicated following therapy with antibiotics, antifungal and antiviral drugs to maintain and restore the balance of vaginal flora.

• Neoprobio is indicated for the prevention and elimination of dry erosion of women of different age groups, as an aid to epithelialization due to the components included in the product.

UNIQUE MEDICAL EFFECTS

Indications are supported by positive clinical trials.

Thank you!

FOR MORE INFORMATION PLEASE CONTACT:

Taras Lyaskovsky, PhDemail: [email protected]

orMichał Szczęsny, MPharm

email: [email protected]

www.neoprobio.pl

vaginal dysbiosis and its correction with vaginal probiotic neoprobio taras lyaskovsky phd,...

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