La microflore vaginale, la vaginose et sa recurrence

Mario VaneechoutteLaboratoire de MicrobiologieHôpital Universitaire de Gand

Flandres, Belgique

RICAI 200626ième Réunion Interdisciplinaire de Chimiothérapie Anti-Infectieuse

Palais des Congrés de Paris, Porte Maillot, Paris, France7-8 décembre 2006

La Microflore Vaginale

Protective role of normal vaginal microflora

Oestrogen

Glycogen

Lactic acid

H2O2 Bacteriocines

Lactobacillus

Mucus

Epithelial cells

Afweer-cellen

Genus Lactobacillus: currently some 80 species

From 1980 onwards: update of the taxonomy of

the L. acidophilus group

La microflore vaginale: les lactobacilles vaginales

L. acidophilus ssL. amylovorusL. amylolyticusL. crispatusL. gallinarumL. gasseriL. inersL. johnsoniiL. kitasatonis

Accurate and rapid identification of cultured lactobacilli:Accurate and rapid identification of cultured lactobacilli:

Baele M, Vaneechoutte M, Verhelst R, Vancanneyt M, Devriese LA, Haesebrouck F.Baele M, Vaneechoutte M, Verhelst R, Vancanneyt M, Devriese LA, Haesebrouck F.

2002. 2002. Identification of Lactobacillus species using tDNA-PCR.

J Microbiol Methods 50: 263-271.

Until 1995 (biochemical identification): L. acidophilus

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 Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. 2004.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.

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

Fréquence (%) des lactobacilles vaginalesdans le vagin en condition saine et en condition perturbé

à base de cultivation + identification par tDNA-PCR

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.

Normal vaginal microflora

Grade I

Ison et al. 2002

Lactobacillus crispatus

L. jensenii - L. gasseri

Grade Ib

Grade Ia

Verhelst et al. BMC 2005

The normal vaginal microflora

Identification génotypique: Bifidobacterium spp.

Normal vaginal microflora?Microscopie : lactobacilles atypiques?

Grade I-like

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 painfulUropathogens: E. coli (approx. 70%), Enterobacteriacae

Enterococcus faecalis, Staphylococcus spp.Increasing drug resistance among uropathogensSequelae: 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).

BV (more general: disturbed vaginal microflora) isassociated with recurrent UTIHooton TM. 2001. IJAA 17: 259-268Gupta 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.

Sequelae of BV = Implications de microflore vaginale perturbé

Results of the culture independent characterization of the vaginal microflora by cloning

Grade I I I II II III III III

Subject code W1 W2 W3 W4 W5 W6 W7 W8

Age 51 34 38 49 41 46 28 44

Species Number of clones 124 118 107 69 72 125 169 70

Lactobacillus crispatus [AF257097] 66.1 99.1

Lactobacillus gasseri [AF243144] 18.5 99.2

Lactobacillus jensenii [AF243159] 0.9

Lactobacillus vaginalis [AF243177] 0.8

Atopobium vaginae [AF325325] 1.4 41.7 36.0 80.5

Gardnerella vaginalis e [M58744] 0.0 0.0 4.1

Lactobacillus iners [Y16329] 84.1 1.4 12.4

Mobiluncus mulieris [AJ427625] 5.6 3.0

Peptostreptococcus anaerobius [L04168] 0.0 1.6 68.6

Peptoniphilus sp. [D14147] 0.0 14.3

Prevotella bivia 91% [L16475] 7.2 22.2

Prevotella buccalis 96.6% [L16476] 1.6 22.2 31.2

Sneathia (Leptotrichia) sanguinegens [L37789] 1.4 1.4 6.4

Uncultured Megasphaera sp. clone [AY271937] 1.4 4.8

Uncultured Actinobacteridae clone 86% [AB089070] 2.9 0.8

Unidentified clone 1 [AY207059] 6.9 4.0

Atopobium rimae [AF292371] 0.8

Fusobacterium nucleatum [AJ006964] 6.5

Peptostreptococcus sp. [AJ277208] 0.8

Pseudoramibacter alactolyticus [AB036761] 0.8

Streptococcus anginosus group [AF104676] 0.0

Treponema sp. clone [AF023055] 0.8

Porphyromonas levi clone 94% [L16493] 1.6

Unidentified clone 2 [AF371910] 2.4 Other species in non-grade I samples

Aerococcus christensenii [Y17318] 3.2 0.0

Anaerococcus tetradius [AF542234] 0.0

Anaerococcus vaginalis [AF542229] 1.4

Bacteroides ureolyticus [L04321] 0.0

Bifidobacterium biavatii (urinalis) [AJ278695] 1.4

Dialister sp. [AF473837] 2.9

Enterococcus faecalis [AJ420803] 1.4

[Leptotrichia amnionii] [AY078425] 1.4

Prevotella bivia [L16475] 0.0 0.0

Prevotella ruminicola 87% [L16476] 4.3

Streptococcus sp. oral strain [AY005041] 8.6

Unidentified clone 3 [AF371693] 6.4

Ureaplasma urealyticum [AF073455] 1.4

Cloning of 16S rRNA genes amplified directly from normal and disturbed vaginal microflora suggests a strong association between Atopobium

vaginae and bacterial vaginosis.What is the role of L. iners?

Nombre de clonesAge

Species (N = 38)

Verhelst et al. BMC Microbiology 2004

Vaginose: Atopobium vaginae

Association with BV and G. vaginalis independently describedFerris MJ, Masztal A, Martin DH. 2004.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 Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. 2004.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. vaginaeBurton JP, Devillard E, Cadieux PA, Hammond J-A , Reid G. 2004Burton 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 PCRfor A. vaginae and G. vaginalis

Grade N A+G+ A+G- A-G+ A-G-

I 112 10 10 20 60

II 26 15 20 35 30

III 10 80 0 10 10

%

Verhelst et al. BMC Microbiology 2004

Microscopy of vaginal lactobacilli

L. crispatus

L. iners A. vaginae

L. gasseri L. crispatus L. jensenii

Bacterial vaginosisNumber of germs

Normal

Symptoms

Lactobacillus

Gardnerellavaginalis

Anaerobes

pH 4.0 - 4.5 pH 5.0 - 6.0

Etiology of Bacterial Vaginosis (BV)

Etiology for BV

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).

Hay P. 2005.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."

Etiology of BVDisturbance of the normal vaginal econiche:

variation in oestrogen levels --> differences in glycogen concentrationmenses --> nutrient (iron) richvaginal douching --> disturbance

Frequency and kind of intercourse: new male sexual partnermore 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

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. 2005Bacterial vaginosis, a disturbed bacterial flora and treatment enigma. APMIS 113:305-316.

This recurrence might be due to the survival of metronidazole or clindamycin resistantbacteria 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 recurrenteRésistance antibiotique ou Biofilm?

Vaginose recurrente: Atopobium vaginae?

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

Possible reason for BV recurrence problem?

•Gram-positive elliptical cocci• Strictly anaerobic• Very fastidious• Recently described (Falsen, 1999)• One case report (PID, 2003)• Metronidazole resistant (?) (2003, 2004)

De Backer, E., R. Verhelst, H. Verstraelen, G. Claeys, G. Verschraegen, De Backer, E., R. Verhelst, H. Verstraelen, G. Claeys, G. Verschraegen, M. Temmerman, and M. Vaneechoutte. 2006. M. Temmerman, and M. Vaneechoutte. 2006. Antibiotic susceptibility of Atopobium vaginae. . BMC Infectious Diseases 2006, 6:51.

 

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

Clindamycin < 0.016 < 0.016 - 0.047

Colistin > 1024 > 1024

Doxycycline 0.19 - 0.75 0.25 - 32

Kanamycin 8 - 16 16 - 32

Linezolid 0.016 - 0.125 0.125 - 0.19

Metronidazole 2 - 256 0.75 - 16

Nalidixic acid > 256  

Penicillin 0.008 - 0.25  

Rifampicin < 0.002 > 256

Vancomycin 1 - 4 0.004 - 0.047

Susceptibilité de A. vaginae et de G. vaginalis

500 mg metronidazole intravaginal = maximal vaginal concentration of 2-10 mg/L

Alper et al. 1985. Obstetr Gynecol 65: 781-784.Mattila et al. 1983. AAC 23: 721-725.

Strain AM AZ BA XM CI CM CO DC KM

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

CCUG 38953T 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

Susceptibilité de Atopobium vaginae

De Backer E, Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Temmerman M, andVaneechoutte M. 2006: Antibiotic susceptibility of Atopobium vaginae. BMC Infect Dis. 6:51.

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

Vaginose recurrenteRésistance antibiotique ou Biofilm?

Swidsinski A, Mendling W, Loening-Baucke V, Ladhoff A, Swidsinski S, Swidsinski A, Mendling W, Loening-Baucke V, Ladhoff A, Swidsinski S, Hale LP, Lochs H. 2005.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)

G. vaginalis – A. vaginae biofilmdémontré à base de FISH avec probes

G. vaginalis et A. vaginae fluorescentes

Lumen

Epithèle vaginale

Biofilm

Grade III: Bacterial vaginosis. Clue cells:Gardnerella vaginalis + Atopobium vaginae

+ anaerobes

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 temporellementles 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 vaginose recurrente: Conclusions/Hypothèses

La microflore vaginale, la vaginose et sa recurrence

Mario.Vaneechoutte@UGent.beLaboratoire de MicrobiologieHôpital 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.

Vaginal probiotic strain(s)

Lactic acidHydrogen peroxideBacteriocins

Argininedeaminase

[Arginine]

Nitric oxide (NO)

Inflammation Polyaminestrimethylamine

BV organisms

Exfoliation - Discharge - Malodor

Adherence competition

Persistent effect

Well-adhering

Resistant tobacteriophages bacteriocins

Biosurfactantproduction

L. jensenii only

Characteristics of a vaginal probiotic preparation

Coaggregationmolecules

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 cancolonize 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 bacilli1892: 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).1892: Lactic acid is primary molecule responsible for low vaginal pH (Döderlein 1892).

1897: Glycogen is direct precursor of lactic acid in the vagina 1897: Glycogen is direct precursor of lactic acid in the vagina

1960: First clinical trial with vaginal probiotic bacteria? (1960: First clinical trial with vaginal probiotic bacteria? (Eschbach, W., Kludas, M. 1960). Eschbach, W., Kludas, M. 1960).

1969: Wylie 1969: Wylie et al.et al.: : L. acidophilusL. acidophilus

1980. Johnson, J. L., C. F. Phelps, C. S. Cummins, J. London, and F. Gasser.1980. Johnson, J. L., C. F. Phelps, C. S. Cummins, J. London, and F. Gasser. Taxonomy of the Taxonomy of the Lactobacillus acidophilusLactobacillus acidophilus group. group. Int. J. Syst. Bacteriol. 30:53–68.Int. J. Syst. Bacteriol. 30:53–68.

1983. Cato, E. P., W. E. C. Moore, and J. L. Johnson. Synonymy of strains of1983. Cato, E. P., W. E. C. Moore, and J. L. Johnson. Synonymy of strains of “ “Lactobacillus acidophilusLactobacillus acidophilus” group A2 (Johnson et al. 1980) with the type strain of” group A2 (Johnson et al. 1980) with the type strain of Lactobacillus crispatus Lactobacillus crispatus (Brygoo and Aladame 1953) Moore and Holdeman 1970. (Brygoo and Aladame 1953) Moore and Holdeman 1970. Int. J. Syst. Int. J. Syst. Bacteriol. 33: 426-428.Bacteriol. 33: 426-428.

1987: 1987: Giorgi Giorgi et al.et al.:: L. crispatus, L. gasseri, L. jensenii L. crispatus, L. gasseri, L. jensenii ((L. delbrueckiiL. delbrueckii group) group)

1995: Andreu 1995: Andreu et al.et al.: 10: 1077-10-1088 cfu of lactobacilli/ml vaginal fluid cfu of lactobacilli/ml vaginal fluid

1999: Antonio 1999: Antonio et al.et al.: : LactobacillusLactobacillus 1086V = 1086V = L. inersL. iners (no growth on Man Rogosa Sharpe) (no growth on Man Rogosa Sharpe)

Clinical trials with vaginal probiotics

Eschbach, W., Kludas, M. 1957. 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.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.Mohler, R., and C. Brown. 1933. Döderlein's bacillus in the treatment of vaginitis. Am J Obstet Gynecol 25:718-723.