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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