Transcript Figure 2

La microflore vaginale,
la vaginose et sa recurrence
Mario Vaneechoutte
Laboratoire de Microbiologie
Hôpital Universitaire de Gand
Flandres, Belgique
RICAI 2006
26ième Réunion Interdisciplinaire de Chimiothérapie Anti-Infectieuse
Palais des Congrés de Paris, Porte Maillot, Paris, France
7-8 décembre 2006
La Microflore Vaginale
Protective role of normal vaginal microflora
H2O2
Lactic acid
Bacteriocines
Lactobacillus
Mucus
Epitheli
al cells
Glycogen
Afweercellen
Oestrogen
La microflore vaginale: les lactobacilles vaginales
Genus Lactobacillus: currently some 80 species
From 1980 onwards: update of the taxonomy of
the L. acidophilus group
L. acidophilus ss
L. amylovorus
L. amylolyticus
L. crispatus
L. gallinarum
L. gasseri
L. iners
L. johnsonii
L. kitasatonis
Until 1995 (biochemical identification): L. acidophilus
Accurate and rapid identification of cultured lactobacilli:
Baele M, Vaneechoutte M, Verhelst R, Vancanneyt M, Devriese LA, Haesebrouck F.
2002. Identification of Lactobacillus species using tDNA-PCR.
J Microbiol Methods 50: 263-271.
tDNA-PCR pattern of vaginal lactobacilli
L. crispatus
L. jensenii
L. gasseri
L. iners
Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De
Ganck C, Temmerman M, Vaneechoutte M. 2004. Cloning of 16S rRNA genes amplified from
normal and disturbed vaginal microflora suggests a strong association between Atopobium
vaginae, Gardnerella vaginalis and bacterial vaginosis. BMC Microbiol 4:16.
Fréquence (%) des lactobacilles vaginales
dans le vagin en condition saine et en condition perturbé
à base de cultivation + identification par tDNA-PCR
Vaginal smears graded by Gram stain
Species
Normal (439)
Disturbed (68)
H2O2 production
Lactobacillus crispatus A
48,3
7,4
+++
Lactobacillus jensenii D
25,3
38,2
++
Lactobacillus gasseri A
23,5
39,7
++
Lactobacillus iners
A
20,5
27,9
+
Lactobacillus vaginalis
11,6
4,4
++
Lactobacillus coleohominis
3,4
1,5
Lactobacillus reuteri
1,4
0,0
++
Lactobacillus fermentum
1,1
1,5
+
Lactobacillus rhamnosus
0,9
4,4
+
Lactobacillus casei
0,9
2,9
Lactobacillus delbrueckii
0,7
1,5
++
Lactobacillus kalixensis
0,2
0,0
Lactobacillus pontis 94%
0,2
0,0
Lactobacillus salivarius
0,2
0,0
+
Lactobacillus mucosae
0,0
2,9
Lactobacillus oris
0,0
2,9
Lactobacillus nagelii
0,0
1,5
Verhelst, R., H. Verstraelen, G. Claeys, G. Verschraegen, L. Van Simaey, C. De Ganck, E. De
Backer, M. Temmerman, and M. Vaneechoutte. 2005. Comparison between Gram stain and culture
for the characterization of vaginal microflora: Definition of a distinct grade that resembles grade I
microflora and revised categorization of grade I microflora. BMC Microbiol. 2005, 5: 61.
The normal vaginal microflora
Grade Ia
Lactobacillus crispatus
Grade Ib
Normal vaginal microflora
Grade I
Ison et al. 2002
L. jensenii - L. gasseri
Verhelst et al. BMC 2005
Normal vaginal microflora?
Microscopie : lactobacilles atypiques?
Grade I-like
Identification génotypique: Bifidobacterium spp.
La Vaginose Bactérienne (VB)
Infectious problems of the female urogenital tract,
shown to be related to disturbance
of normal vaginal microflora
* VB: "lactobacilli deficiency syndrome", nonspecific vaginitis, G. vaginalis vaginitis
Gardnerella vaginalis, Atopobium vaginae, anaerobes,
(Mycoplasma hominis, Ureaplasma urealyticum)
* UTI: Escherichia coli (Gupta et al. 1998, Atassi et al. 2006),
Gram negatives (Chan et al. 1984, Fraga et al. 2005, Osset et al. 2000)
Staphylococcus aureus
* STD: Chlamydia trachomatis
Mycoplasma genitalium
Neisseria gonorrhoeae
Treponema pallidum: syphilis
HPV: cervix carcinoma (BV is co-factor?)
HIV: BV causes increased HIV shedding (Sewankambo et al. 1997)
Herpes Simplex Virus 2
Trichomonas vaginalis
* Yeast vaginitis: Candida albicans (VB protective? (Rodrigues AG ea 1999)
* Group B streptococci: Streptococcus agalactiae neonatal meningitis
UTI in women: les femmes sont plus vulnérables
Cerveau
300 million - 1 milliard de cas par an (Reid 2001. Am J Clin Nutr 73: S437-S443)
one of the most common reasons for women to visit the family physician.
Each episode: on average 6 days of symptoms, often very painful
Uropathogens: E. coli (approx. 70%), Enterobacteriacae
Enterococcus faecalis, Staphylococcus spp.
Increasing drug resistance among uropathogens
Sequelae:
kidney infection (pyelonephritis) --> preterm birth
preterm birth
Bacterial vaginosis (BV):
Symptoms and diagnosis
BV - nonspecific vaginitis - G. vaginalis vaginitis • Prevalence
– 5-35% of Caucasian women
– > 50% of black African women
• Microscopy: clue cells + overgrowth of bacteria
• pH raised from 4-4.5 to 6
• Only 50-60% symptomatically:
– Itch
– Vaginal discharge: due to desquamation of vaginal
epithelial cells (clue cells: covered with bacteria)
– Malodor: due to production by anaerobe bacteria of
polyamines (triethylamine, putrescine, cadaverine)
Sequelae of BV =
Conséquences de microflore vaginale perturbé
BV:
increases HSV2 infection (Cherpes TL 2005. CID 40: 1422).
increases susceptibility for HIV-infection
increases HIV shedding (Cu-Uvin S. 2004. CID 33: 894).
(Sewankambo. 1997. Lancet 350: 546).
--> increases perinatal mother-child HIV-transmission
--> increases sexual HIV-transmission
increases infection with CT and HPV (da Silva CS. 2004. GOInvest. 58: 189).
Sequelae of BV =
Implications de microflore vaginale perturbé
BV (more general: disturbed vaginal microflora) is
associated with recurrent UTI
Hooton TM. 2001. IJAA 17: 259-268
Gupta et al. 1998. Inverse association of H2O2-producing lactobacilli and
vaginal Escherichia coli colonization in women with recurrent urinary tract infections.
J Infect Dis 178: 446-450.
responsible for 30% of PTB
--> 70% of all neonatal mortality & morbidity
(PTB & PTL: 4 billion US$/year)
associated with PID, postpartum endometritis, ...
Jacobsson et al. 2002. Bacterial vaginosis in early pregnancy may predispose for preterm birth
and postpartum endometritis. Acta Obstet Gynecol Scand 81:1006-1010.
cause of asthma?
Benn et al. 2002. Maternal vaginal microflora during pregnancy
and the risk of asthma hospitalization and use of antiasthma medication in early childhood.
J Allergy Clin Immunol 110: 72- 77.
Cloning of 16S rRNA genes amplified directly from normal and disturbed
Results ofsuggests
the culture
independent
characterization
vaginal microflora
a strong
association
between Atopobium
ofvaginae
the vaginal
by cloning
and microflora
bacterial vaginosis.
What is the role of L. iners?
Grade
Subject code
Age
Age
Species
(N
=
38)
Species
Number
of clones
Nombre
de clones
Lactobacillus crispatus [AF257097]
Lactobacillus gasseri [AF243144]
Lactobacillus jensenii [AF243159]
Lactobacillus vaginalis [AF243177]
Atopobium vaginae [AF325325]
Gardnerella vaginalis e [M58744]
Lactobacillus iners [Y16329]
Mobiluncus mulieris [AJ427625]
Peptostreptococcus anaerobius [L04168]
Peptoniphilus sp. [D14147]
Prevotella bivia 91% [L16475]
Prevotella buccalis 96.6% [L16476]
Sneathia (Leptotrichia) sanguinegens [L37789]
Uncultured Megasphaera sp. clone [AY271937]
Uncultured Actinobacteridae clone 86% [AB089070]
Unidentified clone 1 [AY207059]
I
W1
I
W2
51
124
66.1
18.5
I
W3
34
118
II
W4
38
107
99.1
II
W5
III
W6
III
W7
III
W8
49
69
41
72
46
125
28
169
1.4
0.0
84.1
41.7
36.0
0.0
80.5
4.1
12.4
3.0
44
70
99.2
0.9
0.8
1.4
0.0
7.2
1.6
Atopobium rimae [AF292371]
0.8
Verhelst nucleatum
et al. BMC
Microbiology6.52004
Fusobacterium
[AJ006964]
1.4
1.4
2.9
22.2
22.2
1.4
6.9
5.6
1.6
0.0
31.2
6.4
4.8
0.8
4.0
68.6
14.3
Vaginose: Atopobium vaginae
Association with BV and G. vaginalis independently described
Ferris MJ, Masztal A, Martin DH. 2004. Use of species-directed 16S rRNA gene
PCR primers for detection of Atopobium vaginae in patients with bacterial
vaginosis. J Clin Microbiol 42:5892-4.
Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van
Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. 2004. Cloning of 16S
rRNA genes amplified from normal and disturbed vaginal microflora suggests a
strong association between Atopobium vaginae, Gardnerella vaginalis and
bacterial vaginosis. BMC Microbiol 4:16.
Other simultaneous publications on A. vaginae
Burton JP, Devillard E, Cadieux PA, Hammond J-A , Reid G. 2004. Detection of
Atopobium vaginae in postmenopausal women by cultivation-independent
methods warrants further investigation J Clin Microbiol 42: 1829-1831.
Results of species specific PCR
for A. vaginae and G. vaginalis
%
Grade
I
N
112
A+G+ A+G- A-G+
10
10
20
A-G60
II
26
15
20
35
30
III
10
80
0
10
10
Verhelst et al. BMC Microbiology 2004
Microscopy of vaginal lactobacilli
L. iners
L. crispatus L. crispatus
A. vaginae
L. gasseri
L. jensenii
Etiology of Bacterial Vaginosis (BV)
Number
of germs
Bacterial vaginosis
Normal
Lactobacillus
Symptoms
Gardnerella
vaginalis
Anaerobes
pH 4.0 - 4.5
pH 5.0 - 6.0
Etiology for BV
Hay P. 2005. Life in the littoral zone: lactobacilli losing the plot.
Sex Transm Infect. 81:100-102.
"The vagina is not a steady state ecosystem.
As the menstrual cycle becomes established, there are marked cyclical changes
in the vaginal environment, which can be compared to life in the littoral zone of
the seashore:
Levels of oestrogen and progesterone alter,
changing the endogenous environment for lactobacilli by influencing
levels of glycogen and glucose as substrate,
and levels of vaginal pH."
Oestrogen is protective:
BV prevalence lower in women using combined oral contraception (Yen et al. 2003).
BV lower during pregnancy (lower during third trimester) (Hay et al. 1994)
Clinical trials with oestradiol show cure of BV, restoration pH, ...
(Kanne & Jenny 1991, Raz & Stamm 1993, Parent et al. 1996, Ozkinay et al. 2005)
Progesterone treatment induces BV-like microflora in mice
(Furr & Taylor-Robinson 1991).
Etiology of BV
Disturbance of the normal vaginal econiche:
variation in oestrogen levels --> differences in glycogen concentration
menses
--> nutrient (iron) rich
vaginal douching
--> disturbance
Frequency and kind of intercourse: new male sexual partner
more male sexual partners: promiscuity = STD?
female sexual partner
cfr. Vallor et al. 2001: Sexual intercourse once a week was the only risk factor
associated with loss of H2O2 producing lactobacilli.
Again: frequent disturbance of pH by alkaline sperm
--> raise of pH
(Boskey et al. 1993: acidification by lactobacilli takes several hours)
Etiologie de VB: hypothèse/conclusions
• Le risque à développement de VB dépend largement
de la fréquence et de l'intensité de perturbance
de la microflore vaginale lactobacillienne
qui dépend à son tour:
• de la microflore même originelle du vagin:
– des forts vers des faibles protecteurs
• de l'intensité de la menstruation
• de l'age de la femme:
– femmes en menarche sont mieux protégés
par la production des oestrogènes
• de l'intensité de l'acitivité sexuelle
• d'autres practiques perturbants (vaginal douching)
La vaginose recurrente
Vaginose recurrente
Résistance antibiotique ou Biofilm?
Recurrence rates of up to 80% within 3 months after treatment have been reported:
Hay P. 2000. Recurrent bacterial vaginosis. Curr Infect Dis Rep 2:506-512.
Larsson PG & U. Forsum. 2005
Bacterial vaginosis, a disturbed bacterial flora and treatment enigma.
APMIS 113:305-316.
This recurrence might be due to the survival of metronidazole or clindamycin resistant
bacteria in the vagina,
although Beigi et al. [2004] showed that less than one percent
of vaginal anaerobes is metronidazole resistant:
Beigi RH et al. 2004.
Antimicrobial resistance associated with the treatment of bacterial vaginosis.
Am J Obstet Gynecol 191:1124-1129.
Vaginose recurrente:
Atopobium vaginae?
•Gram-positive elliptical cocci
• Strictly anaerobic
• Very fastidious
• Recently described (Falsen, 1999)
• One case report (PID, 2003)
• Metronidazole resistant (?) (2003, 2004)
Metronidazole resistant?
Geissdörfer et al. 2003. J Clin Microbiol 41:2788-2790.
Ferris et al. 2004. Association of Atopobium vaginae, a recently described
metronidazole resistant anaerobe, with bacterial vaginosis.
BMC Infect Dis 4:5:
ensemble: 4 souches testés: MIC metronidazole: > 32 µg/ml
De Backer, E., R. Verhelst, H. Verstraelen, G. Claeys, G. Verschraegen,
Possible
reason forand
BVM.
recurrence
problem?
M. Temmerman,
Vaneechoutte.
2006.
Antibiotic susceptibility of Atopobium vaginae.
BMC Infectious Diseases 2006, 6:51.
Susceptibilité de A. vaginae et de G. vaginalis
Atopobium vaginae
Gardnerella vaginalis
(n=9)
(n=4)
Antimicrobial agent
Range (mg/L)
Range (mg/L)
Ampicillin
< 0.016 - 0.94
< 0.016 - 0.047
Azithromycin
< 0.016 - 0.32
< 0.016 - 0.047
Bacitracin
1-4
0.75 -2
Cefuroxim
0.016 - 0.25
< 0.016 - 0.125
Ciprofloxacin
0.023
- 0.25
0.75 - 2
500 mg metronidazole
intravaginal
=
Clindamycin
< 0.016 of 2-10 mg/L
< 0.016 - 0.047
maximal vaginal concentration
Colistin
> 1024
> 1024
Alper et al. 1985. Obstetr
Gynecol 65: 781-784.
0.19 - 0.75
0.25 - 32
Mattila et al. 1983. AAC 23: 721-725.
Doxycycline
Kanamycin
8 - 16
16 - 32
0.016 - 0.125
0.125 - 0.19
Metronidazole
2 - 256
0.75 - 16
Nalidixic acid
> 256
Linezolid
Penicillin
0.008 - 0.25
Rifampicin
< 0.002
> 256
Vancomycin
1-4
0.004 - 0.047
Susceptibilité de Atopobium vaginae
Strain
AM
AZ
BA
XM
CI
CM
CO
DC
K
M
LZ
MZ
NA
PG
RI
VA
CCUG 42099
0.094
< 0.016
3
0.25
0.064
< 0.016
> 1024
0.75
12
0.094
2
> 256
0.25
< 0.002
2.0
CCUG 44116
0.032
< 0.016
3
0.125
0.25
< 0.016
> 1024
0.19
12
0.032
4
> 256
0.064
< 0.002
1.5
CCUG 44258
0.023
< 0.016
4
0.19
0.064
< 0.016
> 1024
0.38
16
0.023
> 256
> 256
0.094
< 0.002
1.5
PB2003/009-T1-4
< 0.016
< 0.016
1
0.016
0.023
< 0.016
> 1024
0.38
16
0.125
12
> 256
0.008
< 0.002
1.0
PB2003/017-T1-2
< 0.016
< 0.016
1.5
0.125
0.032
< 0.016
> 1024
0.25
16
0.125
> 256
> 256
0.008
< 0.002
1.5
0.094
< 0.016
3
0.023
0.032
< 0.016
> 1024
0.25
8
0,125
> 256
> 256
0.125
< 0.002
1.5
CCUG 44125
0.047
< 0.016
3
0.25
0.064
< 0.016
> 1024
0.19
12
0.047
8
> 256
0.19
< 0.002
1.0
CCUG 44061
0.023
< 0.016
3
0.19
0.047
< 0.016
> 1024
0.38
12
0.023
16
> 256
0.19
< 0.002
1.5
PB2003/189-T1-4
0.016
0.32
3
0.125
0.19
< 0.016
> 1024
0.38
16
0.016
6
> 256
0.008
< 0.002
1.0
CCUG 38953
T
De Backer E, Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Temmerman M, and
Vaneechoutte M. 2006: Antibiotic susceptibility of Atopobium vaginae.
BMC Infect Dis. 6:51.
Vaginose recurrente
Résistance antibiotique ou Biofilm?
L'importance du biofilm dans l'infection:
Public announcement of US National Institute of Health:
"Biofilms are medically important,
accounting for over 80% of microbial infections in the body"
(Davies 2003. Nature Reviews 2: 114-122)
infection of URT in CF-patients: Pseudomonas aeruginosa
chronic otitis media: Haemophilus influenzae, Alloiococcus otitidis?
burn wounds: Pseudomonas aeruginosa, Staphylococcus aureus
foreign object infections: catheters, valves, ...: Staphylococcus spp.
acne: Propionibacterium acnes
recurrent UTI: uropathogenic Escherichia coli
bacterial vaginosis: Gardnerella vaginalis, Atopobium vaginae
G. vaginalis – A. vaginae biofilm
démontré à base de FISH avec probes
G. vaginalis et A. vaginae fluorescentes
Swidsinski A, Mendling W, Loening-Baucke V, Ladhoff A, Swidsinski S, Hale
LP, Lochs H. 2005.
Adherent biofilms in bacterial vaginosis. Obstetrics & Gynecol. 106: 1013-1023.
86% of the G. vaginalis biofilms were associated with Atopobium,
which could reach up to 40% of the biofilm mass (i.e. 4 x 1010 bacteria per mm2)
Lumen
Biofilm
Epithèle vaginale
Grade III: Bacterial vaginosis. Clue cells:
Gardnerella vaginalis + Atopobium vaginae
+ anaerobes
La vaginose recurrente: Conclusions/Hypothèses
Hypothèses:
1. La recurrence peut être causée par des souches résistentes pour la métronidazole.
Bienque 99% des anaerobes soient susceptibles (Beigi et al. 2004),
plusieurs de souches de G. vaginalis et de A. vaginae sont très résistant.
2. La recurrence est probablement surtout la conséquence de la formation du biofilm
par G. vaginalis en association avec A. vaginae.
Traitement de VB avec des antibiotiques fait disparaître temporellement
les symptomes cliniques,
mais le biofilm ne peut pas être éradicé
et résuscite après quelques jours/semaines/mois.
En accordance avec l'apparition des 'clue cells'
La microflore vaginale,
la vaginose et sa recurrence
[email protected]
Laboratoire de Microbiologie
Hôpital Universitaire de Gand
Flandres, Belgique
Presentation downloadable at
http//users.ugent.be/~mvaneech/LBR.htm
Merci pour votre attention
Traitement: probiotiques vs
antibiotiques
Rationale pour la dévéloppement
des probiotiques vaginales
Disturbed vaginal microflora is important: medically and commercially:
Urogenital tract infections are a major problem,
directly (patient) and with consequences (PTB, HIV, STD, ...)
There is a clear hypothesis about role of lactobacilli (<> intestine?):
The protective role of vaginal lactobacilli is clearly established.
Several mechanisms of protective activity have been proposed
and can serve as selection criteria for probiotic strains.
Lactobacilli are predominant in the vagina (<> intestine)
Application can be topical (<> intestine) --> reaching high inocula
Clinical trials are easy to perform (<> intestine):
Sampling is easy during clinical trials.
Re-isolation of probiotic lactobacilli after application is easy.
Several products are already available.
Characteristics of a vaginal probiotic preparation
Vaginal probiotic strain(s)
Coaggregation
molecules
Resistant to
bacteriophages
bacteriocins
Persistent effect
Well-adhering
Arginine
deaminase
Biosurfactant
production
Lactic acid
Hydrogen peroxide
Bacteriocins
Adherence
competition
L. jensenii only
[Arginine]
BV organisms
Nitric oxide (NO)
Inflammation
Polyamines
trimethylamine
Exfoliation - Discharge - Malodor
Les probiotiques vis à vis les antibiotiques:
quelques remarques
Probiotics vs antibiotics?
Antibiotics (clindamycine more than metronidazole) damage commensal microflora
Antibiotics can increase the occurrence of resistant bacteria
Antibiotics can have adverse side effects (especially in pregnant women)
Probiotics can be used in adjunction to antibiotics to restore the commensal microflora
Antibiotics may be needed as initial therapy to increase the chance that probiotics can
colonize the vagina
Boskey et al. 2001: all vaginal products should be tested for
absence of toxicity vs lactobacilli before being admitted.
History of characterization of vaginal
microflora and vaginal probiotics
1892: Döderleins' bacilli: vaginal microflora consists of one kind of Gram-positive bacilli
1892: Lactic acid is primary molecule responsible for low vaginal pH (Döderlein 1892).
1897: Glycogen is direct precursor of lactic acid in the vagina
1960: First clinical trial with vaginal probiotic bacteria? (Eschbach, W., Kludas, M. 1960).
1969: Wylie et al.: L. acidophilus
1980. Johnson, J. L., C. F. Phelps, C. S. Cummins, J. London, and F. Gasser.
Taxonomy of the Lactobacillus acidophilus group. Int. J. Syst. Bacteriol. 30:53–68.
1983. Cato, E. P., W. E. C. Moore, and J. L. Johnson. Synonymy of strains of
“Lactobacillus acidophilus” group A2 (Johnson et al. 1980) with the type strain of
Lactobacillus crispatus (Brygoo and Aladame 1953) Moore and Holdeman 1970.
Int. J. Syst. Bacteriol. 33: 426-428.
1987: Giorgi et al.: L. crispatus, L. gasseri, L. jensenii (L. delbrueckii group)
1995: Andreu et al.: 107-108 cfu of lactobacilli/ml vaginal fluid
1999: Antonio et al.: Lactobacillus 1086V = L. iners (no growth on Man Rogosa Sharpe)
Clinical trials with vaginal probiotics
Eschbach, W., Kludas, M. 1957.
Über die Fortdauer einer Besiedlung der menschlichen Scheide mit
lebenden, lyophilisierten Döderleinbakterien auf Wattetampons.
Ärtzliche Wochenschrift 12: 739-742.
Eschbach, W., Kludas, M. 1960.
On the maintenance and restoration of ideal vaginal states in
sexually mature women by Doederlein bacteria.
Med. Klin. 55: 1114-1116.
Mohler, R., and C. Brown. 1933.
Döderlein's bacillus in the treatment of vaginitis.
Am J Obstet Gynecol 25:718-723.