Probiotics as magic

bullets for vulvovaginal candidiasis?

Leslie Thian Lung, THAN Department of Medical Microbiology and Parasitology,

Faculty of Medicine and Health Sciences, Universiti Putra Malaysia

Source: http://www.travisnation.com/wp-content/uploads/2013/11/no-magic-bullets.jpg

Probiotics Congress: Asia 2016

Selamat datang !!

About VVC… • Vulvovaginal candidiasis (VVC) is a common superficial infection of

the vaginal mucosa membrane caused by Candida species as a result of

unbalanced vaginal microbiome.

• More than 60% of diabetic patients with VVC were caused by C.

glabrata – response poorly to the antifungal treatment (8-fold resistance

to fluconazole). (Goswami et al., 2006)

VVC

Uncomplicated VVC

* C. albicans (>85%)

* Treated effectively with current antimycotic agents

Complicated VVC

* Non-C. albicans Candida species (NCAC)

* Predisposing factors

* Recurrent attack

What’s the fuss with VVC…

Problems of current

treatments in VVC

Drug resistant Candida strains

Increased prevalence of NCAC

species

Drug interaction / adverse

effects

Recurrent VVC

Necessitated

“paradigm shift”

in the therapeutic

or prophylactic

approaches for

complicated VVC

PROBIOTIC

Restore the balance of

vaginal microbiomes!

A little bit of background…

• Probiotics – “live microorganisms which when administrated in adequate

amount confer a health benefit on the host”. (Reid et al., 2003)

• Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14:

• Decrease the number of pathogenic yeast in vaginal cavity.

• Decrease the risk of urogenital diseases such as bacterial vaginosis

(BV) and VVC.

• Improve efficacy of fluconazole treatment. (Reid et al., 2003, Cianci et al., 2008, Martinez et al., 2009)

• The antagonistic effects of these lactobacilli strains have never been studied on other emerging NCAC species (e.g. C. glabrata).

• Our study aimed to provide insights into the probiotic interaction of these lactobacilli strains with the vaginal pathogen C. glabrata.

# SPOT OVERLAY ASSAY

C. glabrata ATCC 2001 C. glabrata 91152 C. glabrata 94885

MR

S

MR

S-M

OP

S, p

H 7

* Note: Colonies on the left – L. rhamnosus GR-1 and colonies on the right – L. reuteri RC-14

C. glabrata

ATCC 2001

L. rh

am

nosu

s G

R-1

L. R

eute

ri R

C-1

4

Ratio of growth inhibition zones

= 𝐃𝐢𝐚𝐦𝐞𝐭𝐞𝐫 𝐨𝐟 𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 𝐳𝐨𝐧𝐞 𝐦𝐦 , 𝐫𝐞𝐝 𝐳𝐨𝐧𝐞

𝐃𝐢𝐚𝐦𝐞𝐭𝐞𝐫 𝐨𝐟 𝐥𝐚𝐜𝐭𝐨𝐛𝐚𝐜𝐢𝐥𝐥𝐢 𝐜𝐨𝐥𝐨𝐧𝐲 𝐦𝐦 , 𝐛𝐥𝐮𝐞 𝐳𝐨𝐧𝐞

# SPOT OVERLAY ASSAY

Lactobacilli strains

(Media)

C. glabrata strains (Ratio of growth inhibition zones )

Reference Clinical

ATCC 2001 2737 2744 91152 94885 95670 98328

L. rhamnosus GR-1

(MRS) 1.63 ± 0.04 1.36 ± 0.02 1.33 ± 0.04 1.33 ± 0.03 1.43 ± 0.14 1.37 ± 0.02 1.43 ± 0.03

L. rhamnosus GR-1

(MRS-MOPS) 1.48 ± 0.03 1.15 ± 0.04 1.24 ± 0.01 1.18 ± 0.04 1.19 ± 0.05 1.16 ± 0.08 1.22 ± 0.06

L. reuteri RC-14

(MRS) 1.54 ± 0.05 1.46 ± 0.00 1.38 ± 0.05 1.39 ± 0.07 1.52 ± 0.09 1.35 ± 0.06 1.50 ± 0.03

L. reuteri RC-14

(MRS-MOPS) 1.43 ± 0.03 1.11 ± 0.01 1.19 ± 0.02 1.18 ± 0.00 1.20 ± 0.05 1.26 ± 0.07 1.24 ± 0.03

Ratio of growth inhibition zones of probiotic lactobacilli strains against C. glabrata on MRS and MRS-MOPS agar. Results

shown were the mean of triplicate from three independent experiments ± SD

# PLATE-BASED MICROTITRE ASSAY C

. gla

bra

ta

AT

CC

2001

C

. gla

bra

ta

94

88

5

Treated with FCS Treated with neutralised FCS

Note: ●: FCS produced by L. rhamnosus GR-1, ■ FCS produced by L. reuteri RC-14 and ▲: MRS broth (untreated

control). Results shown were the mean of triplicate from three independent experiments ± SD

~70% inhibition

~50% inhibition

~15% inhibition

~15% inhibition

# GROWTH INHIBITION ACTIVITY

• Both lactobacilli strains inhibited the growth of C. glabrata ATCC 2001

and other clinical isolates of C. glabrata.

• Mainly contributed by the low/ acidic pH – production of organic acids

(e.g. lactic acid bacteria - lactic acid).

• Other inhibitory mechanisms could play some roles in the growth

inhibition observed.

Nutrient

competition (Deriu et al., 2013)

Bacteriocin (Cleusix et al., 2007)

Hydrogen

peroxide (Pridmore et al., 2008)

Biosurfactant (Gudina et al., 2010)

• L. rhamnosus GR-1: non-H2O2

producer

• C. glabrata is highly resistant to

H2O2 (production of catalase)

# FLUORESCENCE CELL VIABILITY ASSAY

C. glabrata ATCC 2001 C. glabrata 94885

Note: ▼: Challenged by L. rhamnosus GR-1, ♦: Challenged by L. reuteri RC-14, ▲:

Monospecies C. glabrata only (untreated control), ●: Monospecies L. rhamnosus GR-1 only and

■: Monospecies L. reuteri RC-14 only. Results shown were the mean of triplicate from three

independent experiments ± SD

Viable C.

glabrata cells

Viable C.

glabrata cells

# CONFOCAL LASER SCANNING

MICROSCOPY (CLSM)

C. glabrata ATCC 2001 (untreated control)

Calcofluor white M2R FUN-1 Stain

Zoomed area of the CLSM image indicated orange-red cylindrical intravacuolar

structures (CIVS) formation (white arrows) – viable cells

# CONFOCAL LASER SCANNING

MICROSCOPY (CLSM)

C. gla

bra

ta A

TC

C 2

001

Calcofluor white M2R FUN-1 Stain

No viable C. glabrata cells

(No CIVS formation, diffused

green yellow fluorescence)

L. rhamnosus GR-1

treated

L. rhamnosus GR-1

treated

L. reuteri RC-14

treated

L. reuteri RC-14

treated

# CANDIDACIDAL ACTIVITY

• Both lactobacilli strains demonstrated strong

candidacidal activity against C. glabrata (fungicidal).

• The present of lactobacilli strains appeared to shut

down the metabolic activity of C. glabrata

completely (no orange-red CIVS formation).

• Formation of multicellular lactobacilli aggregates – crucial for

colonisation and adhesion of the cells on mucosa surface. (Kos et al., 2003)

• Strong autoaggregation phenotype (L. reuteri RC-14 – superior strain in

term of autoaggregation).

# AUTOAGGREGATION ASSAY

Different alphabets in the same columns indicate statistical significance (P < 0.05). Results

shown were the mean of triplicate from three independent experiments ± SD

• Formation of coaggregates - create a hostile niche for the pathogens and prevent colonisation of these microorganisms. (Younes et al., 2012)

• Strong coaggregation phenotype (L. reuteri RC-14).

# COAGGREGATION ASSAY

Different alphabets indicate statistical significance (P < 0.05). Results shown were the

mean of triplicate from three independent experiments ± SD

Results shown were the mean of triplicate from three independent experiments ± SD

• MATH test – measure cell surface hydrophobicity – hydrocarbons

xylene and toluene. (Kos et al., 2003, Ekmekci et al., 2009)

• Strong aggregation phenotypes are usually associated with high

hydrophobicity - contributed by the nature of cell surface components. (Cuperus et al., 1995, Pelletier et al., 1997)

# MICROBIAL ADHESION TO

HYDROCARBONS (MATH)

High hydrophobicity

(Glycol-) proteinaceous

compounds

Low hydrophobicity

Polysaccharides compounds

Hydrophobic strain

Hydrophilic strain

# XTT REDUCTION ASSAY

(A) C. glabrata ATCC 2001, (B) C. glabrata 2737, (C) C. glabrata 2744, (D) C. glabrata 91152, (E) C. glabrata 94885,

(F) C. glabrata 95670, (G) C. glabrata 98328. Results shown were the mean of triplicate from three independent

experiments ± SD. * P < 0.05 was considered statistically significant when compared with untreated controls.

# SCANNING ELECTRON MICROSCOPY

C. g

labra

ta A

TC

C 2

001

• Profused growth and dense colonisation on the coverslips.

• Comprised only of multilayer dense yeast cells and blastoconidia.

# SCANNING ELECTRON MICROSCOPY

C. g

labra

ta A

TC

C 2

001

L. rhamnosus GR-1 treated L. rhamnosus GR-1 treated

L. reuteri RC-14 treated L. reuteri RC-14 treated

Reduction of

biofilms

densities

Deformed

morphologies

and cell surface

structures

RESULTS AND DISCUSSIONS # SCANNING ELECTRON MICROSCOPY

C. g

labra

ta 9

4885

L. rhamnosus GR-1

treated

L. reuteri RC-14

treated Untreated control

# GENE EXPRESSION STUDIES

Gene expression studies

Biofilm-related genes (EPA6 and YAK1)

Key enzyme genes in alternative carbon

utilisation pathways (ICL1 and PCK1)

Drug efflux genes (CDR1 and PDH1)

The impact of lactobacilli challenge on selected C. glabrata genes

were investigated by using quantitative real-time PCR.

# GENE EXPRESSION STUDIES

Relative expression of C. glabrata EPA6 and YAK1 genes were determined quantitatively following

FCS treatment. The housekeeping gene ß-actin, ACT1 was used for normalisation of real-time PCR

data. Results shown were the mean of triplicate from three independent experiments ± SD. * P < 0.05

was considered statistically significant when compared with untreated controls.

# GENE EXPRESSION STUDIES

Relative expression of C. glabrata ICL1 and PCK1 genes were determined quantitatively following

FCS treatment. The housekeeping gene ß-actin, ACT1 was used for normalisation of real-time PCR

data. Results shown were the mean of triplicate from three independent experiments ± SD. * P < 0.05

was considered statistically significant when compared with untreated controls.

# GENE EXPRESSION STUDIES

Relative expression of C. glabrata CDR1 and PDH1 genes were determined quantitatively following

FCS treatment. The housekeeping gene ß-actin, ACT1 was used for normalisation of real-time PCR

data. Results shown were the mean of triplicate from three independent experiments ± SD. * P < 0.05

was considered statistically significant when compared with untreated controls.

Candida glabrata

CONCLUSION

Lactobacillus rhamnosus GR-1

Lactobacillus reuteri RC-14

• Fungicidal

• Biofilms inhibition

• Strong aggregation phenotype

• Down-regulation of C. glabrata genes related to

virulence and survivability.

antagonise

Potential use of these probiotic strains as the main or alternative therapeutic or

prophylactic options for patients with complicated VVC (C. glabrata).

Purification and identification of

antifungal compounds

Mechanism of probiotic actions

In vivo murine model and

clinical trials

What’s

next..??

Some issues to be pondered upon..

Yes…we see effects in in vitro…so what?

1. There is complex profiles in VVC patients (Liu et al. 2013, PLOS One).

2. Our genetics influenced the outcome of our microbial phenotype. (Goodrich et al. 2014, Cell).

3. Mode of delivery, dose and formulations? 4. and etc.

VVC varying microbiota profiles..!!

(Liu et al. 2013, PLOS One)

Probiotics in vitro investigations… (Matsubara et al 2016., CID)

Probiotics in vitro investigations… (Matsubara et al 2016., CID)

Probiotics in vitro investigations… (Matsubara et al 2016., CID)

Probiotics clinical investigations… (Matsubara et al 2016., CID)

The way forward is the multi-omics approach…

http://users.metu.edu.tr/bicgen/research/images/metag.jpg

Immunomodulation study

Take home messages

http://www.shoot.lv/wp-content/uploads/2012/05/photo-black-silhouette-targets-contact-us.jpg

• Though we see strong inhibitory effects of probiotics on C. glabrata in in vitro settings, its efficacy on patients needs to be further expanded. • There are several issues that need to be addressed. • Using the new technologies such as the multi-omics approaches, we shall march forward with more confidence in claiming probiotics as the ‘future’.

Probiotics as magic bullets for

vulvovaginal candidiasis…??

Acknowledgement

• Shu Yih Chew • Chr. Hansen A/S • Kee Peng Ng • Yoke Kqueen Cheah • UPM Research

University Grant Scheme (RUGS6),

(04-01-12-1609RU) • Mycology Lab

members, Dept. of Medical Microbiology and Parasitology, UPM.

Terima Kasih | Thank You